Benzathine analogs

ABSTRACT

A compound, or a pharmaceutically acceptable salt or ester thereof, having a structure of: 
     
       
         
         
             
             
         
       
         
         
           
             wherein X is a divalent linking moiety; and 
             R 1 -R 10  are each individually H, optionally-substituted alkyl, optionally-substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, halogen, amino, or hydroxy, provided that at least one of R 3  or R 8  is an optionally-substituted alkyl, a substituted alkoxy, optionally-substituted aryl, optionally-substituted cycloalkyl, optionally-substituted heterocyclic, or halogen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/138,137, filed Apr. 25, 2016, which is a continuation of U.S.application Ser. No. 14/406,189, filed Dec. 5, 2014, now U.S. Pat. No.9,359,284 issued Jun. 7, 2016, which is the U.S. National Stage ofPCT/US2013/030995, filed on Mar. 13, 2013, which claims the benefit ofU.S. Provisional Application No. 61/657,423, filed Jun. 8, 2012, all ofwhich are herein incorporated by reference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.HL116472, HL068135, HL081784, HL096376, HL098174, and HL097376 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Inflammatory disorders underlie numerous human diseases characterized bya highly activated immune system that leads to secretion of largeamounts of circulating pro-inflammatory cytokines after infection withvirulent pathogens, in response to host cell injury, or relatedirritants that activate receptors on immune effector cells (T-cells,macrophages, etc.). For example, sepsis results in over 500,000 deathsin the US each year and pneumonia is the leading cause of death frominfections. Further, noninfectious illnesses (colitis, arthritis) canalso involve cytokines as major mediators of disease pathogenesis. Acentral feature of these infectious disorders is the burst in cytokinerelease, i.e. cytokine storm, from pro-inflammatory cells includingmacrophages, lymphocytes, and PMNs. Under many conditions, the cytokinestorm is exaggerated (hypercytokinemia) and results in a fatal immunereaction with constant activation of immune effector cells that producesustained and supraphysiologic levels of cytokines including TNFα, IL-β,and IL-6 that leads to profound tissue injury. Left, unchecked, thisprofound inflammatory cascade can have devastating consequences for thehost.

Prior efforts on blocking the cytokine storm has focused on the use ofsystemic corticosteroids or the development of targetedanti-inflammatory agents to specific cytokines, e.g. TNFα and IL-1β thathave not improved mortality in sepsis. Other approaches focusing oninhibiting upstream surface receptors within T-cells (e.g. TLR4receptor) have been inconclusive and similar agents have not succeededin Phase 3 clinical trials. Many of these approaches are limited as onlyone target (a receptor or cytokine) is selected for inhibition; however,systematic inflammation and sepsis are intricate disorders whereby amultitude of inflammatory mediators are released from activation ofmultiple receptors. Agents that are directed against a single moleculartarget cannot prevent activities of other pro-inflammatory cytokinesduring the host inflammatory response. These observations underscore theimportance of identifying newer targets for intervention that mightgovern the synthesis and secretion of a wider array of pro-inflammatorybiomolecules. Further, the mainstay of therapies for sepsis isantimicrobial agents that do not provide total protection and arelimited because of attendant toxicities and the rapid emergence ofmulti-drug resistance. Thus, the discovery of newer small moleculeanti-inflammatory therapeutics with novel targets could have a profoundimpact on the severity of inflammatory illness such as sepsis.

TNF receptor associated factors (TRAFs) are a family of proteinsprimarily involved in the regulation of inflammation, antiviralresponses, and apoptosis. Six well-characterized TRAF proteins (TRAF1-6)exist and a newer homologue TRAF7 was recently identified. All TRAFmembers share a highly conserved C-terminal domain that mediatesinteractions with transmembrane TNF receptors. Identification of TRAFproteins has contributed significantly to the elucidation of themolecular mechanisms of signal transduction emanating from the TRAFsuperfamily and the Toll like/interleukin-1 receptor (TLR/IL-1R) family.TRAF family proteins interact with the IL-1 receptor, TLRs, CD40, RANK,I-TAC, p75 NGF receptor, etc. Specifically, TRAF2, TRAF5, and TRAF6serve as adapter proteins that link cell surface receptors withdownstream kinase cascades, which in turn activate key transcriptionfactors, such as nuclear factor κB (NFκB), resulting in cytokine geneexpression. With an exaggerated immune response, TRAF-mediated cytokinerelease leads to profound effects of edema, multi-organ failure andshock. The TRAF proteins, however, have a central role as they mediatesignal transduction to elicit transcriptional activation of severaldownstream cytokines. These findings suggest that maneuvers designed toselectively modulate the abundance of TRAF proteins might serve as anovel strategy for therapeutic intervention. However, to date, verylittle is known regarding the molecular regulation of the TRAF family atthe level of protein stability. Strategies directed at modulation ofTRAF protein concentrations in cells might serve as the basis for thedesign of a new class of anti-inflammatory agents.

Ubiquitination of proteins brands them for degradation, either by theproteasome or via the lysosome, and regulates diverse processes. Theconjugation of ubiquitin to a target protein is orchestrated by a seriesof enzymatic reactions involving an E1 ubiquitin-activating enzyme,ubiquitin transfer from an E1-activating enzyme to an E2-conjugatingenzyme, and last, generation of an isopeptide bond between thesubstrate's ε-amino lysine and the c-terminus of ubiquitin catalyzed bya E3-ubiquitin ligase. Of the many E3 ligases, the Skp-Cullin1-F box(SCF) superfamily is among the most studied. The SCF complex has acatalytic core complex consisting of Skp1, Cullin1, and the E2ubiquitin-conjugating (Ubc) enzyme. The SCF complex also contains anadaptor receptor subunit, termed F-box protein, that targets hundreds ofsubstrates through phosphospecific domain interactions. F-box proteinshave two domains: an NH2-terminal F-box motif and a C-terminalleucine-rich repeat (LRR) motif or WD repeat motif. The SCF complex usesthe F-box motif to bind Skp1, whereas the leucine-rich/WD repeat motifis used for substrate recognition.

SUMMARY

One embodiment disclosed herein is a compound, or a pharmaceuticallyacceptable salt or ester thereof, having a structure of formula II:

wherein X is a divalent linking moiety; and

R¹-R¹⁰ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy, provided that at least one of R³ or R⁸ is anoptionally-substituted alkyl, a substituted alkoxy,optionally-substituted aryl, optionally-substituted cycloalkyl,optionally-substituted heterocyclic, or halogen.

Further disclosed herein is a compound, or a pharmaceutically acceptablesalt or ester thereof, having a structure of formula III:

formula IV:

wherein X is a divalent linking moiety; and

R²-R⁵ and R⁷-R¹⁰ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy.

Another embodiment disclosed herein is a method for inhibitingpro-inflammatory cytokine release in a subject, comprising administeringto the subject an FBXO3 inhibitor.

A further embodiment disclosed herein is a method for treating aninflammatory disorder in a subject, comprising administering to thesubject a therapeutically effective amount of an FBXO3 inhibitor.

An additional embodiment disclosed herein is a method for inhibitingFBXO3-induced ubiquitination and degradation of FBXL2, comprisingcontacting FBXO3-containing tissue or cells with a benzathine compound,an optionally-substituted diaminoalkane, a substituted quinoline,haematoxylin, tetramethylenebis, naphthacaine, ampicillin, or elliptine.

Another embodiment disclosed herein is a method for inhibiting bacterialgrowth in a subject or a surface of an object, comprising administeringto the subject or the surface of the object an effective amount of anFBXO3 inhibitor.

A further embodiment disclosed herein is a method for inhibiting abioactivity of FBXO3 protein, comprising contacting FBXO3 with acompound that interacts with amino acid residues Y308, N335, E341, T368and S370 that are present in an ApaG domain cavity of the FBXO3 protein.

The foregoing and will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E. FBXL2 targets TRAFs for polyubiquitination.

FIG. 1A. Immunoblotting showing levels of TRAFs and negative controlproteins, after control (CON) plasmid or ectopic FBXL2 plasmidexpression. FIG. 1B. Cells were transfected with an inducible FBXL2plasmid under control of exogenous doxycycline. Cells were treated withdoxycycline for various times, cells were then collected and celllysates were analyzed for FBXL2, TRAFs, and β-actin by immunoblotting.FIG. 1C. Endogenous FBXL2 was immunoprecipitated and followed by TRAF1-6 immunoblotting. FIG. 1D. In vitro ubiquitination assays. PurifiedSCF complex components were incubated with individual V5-TRAFs and thefull complement of ubiquitination reaction components (second lane fromleft) showing polyubiquitinated TRAF proteins. FIG. 1E. Half-life ofeach TRAF protein with or without FBXL2 overexpression is shown.

FIGS. 2A-2D. FBXL2 is polyubiquitinated at the Lysine 201 site. FIGS.2A-C. Several deletion (FIGS. 2A, 2B) and point (FIG. 2C) mutants ofFBXL2 were designed and cloned into a pcDNA3.1D/V5-HIS vector (upperpanel). Plasmids encoding FBXL2 mutants were transfected into cellsfollowed by MG132 treatment. Cells were collected and cell lysates wereanalyzed for V5-FBXL2 and β-actin by immunoblotting after exposure ofcells to vehicle (lower left) or to MG132 (lower right). FIG. 2D.Half-life studies of wild-type (WT) FBXL2 and FBXL2 K201R.

FIGS. 3A-3K. FBXL2 is phosphorylated and targeted by the SCF E3 ligaseFBXO3 at residue T404. FIG. 3A. Scheme of potential phosphorylationsites within FBXL2 (GPS2.1 prediction). FIG. 3B. Endogenous FBXL2 wasimmunoprecipitated and followed by phospho-threonine immunoblotting.FIG. 3C. Endogenous FBXL2 was immunoprecipitated and followed byimmunoblotting for several candidate kinases. FIG. 3D. Endogenous FBXL2was immunoprecipitated and followed by FBXO3 immunoblotting. FIG. 3E. Invitro ubiquitination assays. Purified SCFFBXO3 complex components wereincubated with V5-FBXL2 and the full complement of ubiquitinationreaction components (right lane) showing polyubiquitinated FBXL2. FIG.3F. Cells were transfected with his tagged FBXL2 deletion mutantplasmids, followed by his-pull down; FBXO3 protein bound to the cobaltbeads was eluted and then resolved in SDS-PAGE followed by FBXO3immunoblotting. FIG. 3G. Half-life studies of WT FBXL2 and FBXL2C-terminal deletion mutants. FIG. 3H. GSK3β consensus sequence withinFBXL2. FIG. 3I. Cells were transfected with plasmids encoding eitherV5-WT FBXL2 or V5-FBXL2 T404A point mutants, transfected cells were thensubjected to immunoprecipitation with V5 antibody followed byphospho-threonine immunoblotting. FIG. 3J. In vitro ubiquitinationassays. Purified SCFFBXO3 complex components were incubated with V5tagged WT FBXL2 or the FBXL2 T404A mutant and the full complement ofubiquitination reaction components showing polyubiquitinated FBXL2(second lane from left). FIG. 3K. Model of FBXO3 targeting FBXL2.

FIGS. 4A-4F. FBXO3 contains a natural occurring mutation at V220. FIG.4A. SNP analysis of FBXL2 protein indicating a V220I mutation. FIG. 4B.Genomic DNA was first extracted from PBMC cells from twenty healthyCaucasian donors followed by SNP genotyping using a TaqMan® SNP probewith real-time PCR. FIG. 4C. Three WT PBMC cells samples and three PBMCcells containing the heterozygous V220I mutation were treated with 2ug/ml of LPS for 24 h before assays for cytokine release using a humancytokine array (R&D). FIG. 4D. In vitro ubiquitination assays. PurifiedSCFFBXO3 or SCFFBXO3V220I mutant complex components were incubated withV5-FBXL2 and the full complement of ubiquitination reaction componentsshowing levels of polyubiquitinated FBXL2. FIG. 4E. Cells weretransfected with V5-WT FBXO3 or the V5-FBXO3V220I mutant plasmids,followed by immunoblotting for V5, FBXL2, and TRAF proteins. FIG. 4F.U937 cells were treated with LPS for an additional 24 h before assayingfor cytokine secretion using a human cytokine array (R&D).

FIGS. 5A-5I. FBXO3V220I is a loss-of-function mutant of FBXO3 in vivo.Lentiviral FBXO3 gene transfer augments the severity of P.aeruginosa-induced lung inflammation and injury. C57BL/6J mice wereadministered intratracheal (i.t.) Lenti-LacZ, Lenti-FBXO3 orLenti-FBXO3V220I (107 CFU/mouse) for 120 h, and 4 mice/group wereinoculated with P. aeruginosa (PA103, 104 PFU/mouse) for 24 h. Mice weremonitored on a FlexiVent to measure lung mechanics (FIGS. 5A-5D). Micewere then sacrificed and lungs were lavaged with saline, harvested, andthen homogenized; lavage protein, cell counts, and cytokine secretionwere determined in (FIGS. 5E-5F, 5I). FIG. 5G. H&E staining wasperformed on lung samples in (FIG. 5A). FIG. 5H. Survival studies ofmice administered i.t. PA103 (105 PFU/mouse, 7 mice per group) wasdetermined. Mice were carefully monitored over time; moribund,preterminal animals were immediately euthanized and recorded asdeceased. Kaplan-Meier survival curves were generated using Prismsoftware.

FIGS. 6A-6I. FBXO3 knockdown ameliorates pseudomonas induced lunginjury. Lentiviral FBXO3 knockdown attenuates the severity of P.aeruginosa-induced lung inflammation and injury. C57BL/6J mice wereadministered i.t. Lentivirus encoding control (CON) shRNA or Lenti-FBXO3shRNA (107 CFU/mouse) for 120 h, and 4 mice/group were inoculated withPA103 (104 PFU/mouse) for 24 h. Mice were monitored on FlexiVent tomeasure lung mechanics (FIGS. 6A-6D). Mice were then sacrificed andlungs were lavaged with saline, harvested, and then homogenized. Lavageprotein, cell counts, and cytokine secretion, were measured in (FIGS.6E, 6F, 6H). FIG. 6G. H&E staining was performed on lung samples in(FIG. 6A). FIG. 6I. Survival study of mice administered i.t. with PA103(105 PFU/mouse, 6 mice per group) was determined. Mice were carefullymonitored over time; moribund, preterminal animals were immediatelyeuthanized and recorded as deceased. Kaplan-Meier survival curves weregenerated using Prism software.

FIGS. 7A-7F. FBXO3 structural analysis reveals a bacterial-like ApaGdomain. FIG. 7A. Several deletion mutants of FBXO3 were designed andcloned into a pcDNA3.1D/V5-HIS vector. FIG. 7B. In vitro ubiquitinationassays. Purified SCFFBXO3 full-length (FL) or truncated FBXO3 proteinswere incubated with V5-FBXL2 and the full complement of ubiquitinationreaction components showing polyubiquitinated FBXL2 (second lane fromleft). FIG. 7C. Structural analysis of the FBXO3-ApaG domain. FIGS.7D-7F. Docking study of the candidate compound, benzathine, interactingwith the FBXO3-ApaG domain.

FIGS. 8A-8D. Generation of FBXO3 inhibitors and docking analysis. FIG.8A-8D. General theme of synthesizing benzathine analogs. Briefly, thetarget benzathine analogs were prepared from benzaldehyde derivativesand diamine derivatives such as ethylenediamine. In general, therelevant benzaldehyde derivatives (0.02 mol) were added to a solution ofethylenediamine (0.01 mol, ˜700 ul) in anhydrous ethanol (20 ml). Theresulting solution was refluxed and stirred for 60 min until theprecipitation of the relevant Schiff base. The Schiff bases werefiltered and washed with cold ethanol. The Schiff base was then added to30 ml absolute methanol. A 10% solution of sodium borohydride (0.02 mol)was dissolved in absolute methanol and added to the Schiff base. Whenthe addition was complete, the reaction solution was refluxed for anadditional 15 min. Solvent was then removed through rotary evaporationand 40 ml cold water was added to liberate the secondary amine. Theprecipitation of benzathine derivatives were collected, washed withwater and dried, followed by recrystallization from ethyl acetate. FIGS.8B-8D. Structure and docking studies of the novel FBXO3 inhibitor,BC-1215.

FIG. 9. BC-1215 inhibits a broad spectrum of Th1 panel cytokines. PBMCcells (0.6 ml at 1.5*10^6/ml) were treated with 2 ug/ml LPS for 16 hiswith BC-1215 at 10 ug/ml. Cytokine release was monitored by the humancytokine array (R&D systems). The results from cytokine array dot blotwere quantitated and graphed in below.

FIGS. 10A-10E. BC-1215 inhibits FBXO3 and decreases TRAF protein levels.FIG. 10A. PBMC cells were treated with 2 ug/ml of LPS at each time pointbefore immunoblotting for indicated proteins. FIG. 10B. In vitroubiquitination assays. Purified SCFFBXO3 complex components wasincubated with V5-FBXL2 and the full complement of ubiquitinationreaction components with increased concentration of BC-1215 showingdecreased levels of polyubiquitinated FBXL2. FIG. 10C. MLE cells werealso treated with BC-1215 at different concentrations for 16 h. Cellswere collected and assayed for immunoblotting.

FIG. 10D. Hela cells were treated with BC-1215 at differentconcentrations for 24 h before cell cycle analysis (BD bioscience). FIG.10E. MLE cells were treated with BC-1215 (10 ug/ml) for 24 h beforeassaying for COX-2 activity (Cayman).

FIG. 11. BC-1215 inhibits cytokine release from in an endotoxin septicshock model. BC-1215 was solubilized in water using acetic acid in a 1:2molar ratio; the stock solution of BC-1215 was 5 mg/ml. C57BL6 mice weredeeply anesthetized with ketamine (80-100 mg/kg intraperitoneally (i.p.)and xylazine (10 mg/kg i.p.). 500 ug, 100 ug, 20 ug, 4 ug and 0.8 ug ofBC-1215 was administered to mice through an intraperitoneal (IP)injection. 10 min later, mice were given 100 ug of LPS (E. coli) throughan IP injection. 90 min later, mice were euthanized; blood was collectedand tested for IL1-β, IL-6 and TNFα assays. (n=3/mice group at eachdose) FIG. 12. BC-1215 inhibits cytokines release in a cecal ligationand puncture (CLP) sepsis model. BC-1215 was solubilized as above.C57BL6 mice were deeply anesthetized with ketamine (80-100 mg/kg IP andxylazine (10 mg/kg i.p.). 100 ug of BC-1215 was administered to micethough an IP injection. 30 min later, CLP was performed. 6 h later, micewere euthanized; blood was collected and assayed for IL1-β, IL-6 andTNFα levels. (n=4-5 mice/group, *p<0.05 versus CLP)

FIGS. 13A-13H. BC-1215 reduces lung injury in pseudomonas pneumonia.BC-1215 (100 ug) was administered to C57BL6 mice though an IP injection,mice were then challenged with Pseudomonas (strain PA103, 104 CFU/mouse,i.t.) for an additional 18 h. Mice were monitored on a FlexiVent tomeasure lung mechanics (FIGS. 13A-13D). Mice were then sacrificed andlungs were lavaged with saline, harvested, and then homogenized. Lavageprotein, cell count and cytokine secretion, was measured in (FIG. 13E,13F, 13H). FIG. 13G. H&E staining was performed on lung samples. (n=4-6mice/group, *p<0.05 versus Vehicle)

FIGS. 14A-14H. BC-1215 lessens severity of H1N1 Influenza pneumonia.FIGS. 14A-14D. C57BL6 mice were challenged with H1N1 (106 PFU/mouse,i.t.) for up to 9 d. For BC-1215 treatment, a stock solution (5 mg/ml)was added to drinking water (containing 2% sucrose) to the finalconcentration of 30 ug/ml. Lung mechanics were measured at day 5 usingFlexiVent (FIGS. 14A-14C). FIG. 14D. Survival study of mice administeredi.t. with H1N1 (10⁵ PFU/mouse, 8 mice/group). Mice were carefullymonitored over time; moribund, preterminal animals were immediatelyeuthanized and recorded as deceased. Mice were then sacrificed and lungswere lavaged with saline, harvested, and then homogenized. Lavageprotein, cell count were measured in (FIG. 14E, 14F). FIG. 14G.Photograph of lungs from vehicle or BC-1215 treated mice. FIG. 14H. H&Estaining was performed on lung samples. (n=5-8 mice/group, *p<0.05versus H1N1)

FIGS. 15A-15C. BC-1215 reduces TPA induced ear edema. FIG. 15A-15C.C57BL6 mice were deeply anesthetized with ketamine (80-100 mg/kg i.p.)and xylazine (10 mg/kg i.p.). 20 μl of ethanol solution of BC-1215 wasapplied to ears at 8, 40, 200 ug/ear 30 min after TPA administration (2μg/ear). Comparisons included equal volumes of ethanol (vehiclecontrol). 18 h after TPA administration, mice were euthanized; thethickness of the ear was measured using a micrometer (FIG. 15B). Earpunch biopsies were also taken immediately, weighed and graphed (FIG.15C).

FIGS. 16A-16C. BC-1215 reduces Carrageenan induced paw edema. FIGS.16A-16C. C57BL6 mice were deeply anesthetized with ketamine (80-100mg/kg i.p.) and xylazine (10 mg/kg i.p.). Mice were received asubplantar administration of 25 ul of saline or 25 ul of carrageenan (1%in saline), followed by an IP injection of 100 ug of BC-1215 daily fortwo days. Mice were then euthanized; the thickness and volume of paw wasmeasured (FIG. 16B-16C). (n=4 mice/group, *p<0.05 versus vehicle) FIGS.17A-17D. BC-1215 reduces DSS induced colonic inflammation. FIGS.17A-FIG. 17C. C57BL6 mice were fed with water containing 3.5% dextransulfate sodium (DSS) for up to five days. Mice were treated with eithervehicle or 100 ug of BC-1215 daily (via IP injection). Mice were theneuthanized; the length of the colon was measured and graphed in (FIG.17A). Colonic tissues were also analyzed for IL1β (FIG. 17B) and TNFα(FIG. 17C) by ELISA. (n=4 mice/group, *p<0.05 versus DSS) FIG. 17D, H&Estaining was performed on colonic samples. (n=4 mice/group, *p<0.05versus DSS)

FIG. 18. A proposed novel inflammatory pathway catalyzed by FBXO3.Infection or autoimmune disorders might involve the following pathway:FBXO3┤FBXL2┤TRAFs→ cytokine production→ tissue inflammation, injury, andedema. Specifically, local and systemic inflammation is regulated inpart, by a unique pathway whereby a previously unrecognized E3 ligasecomponent, FBXO3, triggers ubiquitination and degradation of another E3ligase subunit, FBXL2, thereby increasing levels of TRAF proteins thatmediate cytokine secretion from inflammatory cells. In essence, FBXL2appears to be a feedback inhibitor of inflammation. As TRAFs arecritical molecular inputs to cytokine gene expression via NF-κ, mutationor inhibition of FBXO3 will prevent induction of TRAF proteins andsuppress cytokine production. FBXO3 serves as a novel molecular targetas the centerpiece of this invention that has led to the genesis of Fbox protein ubiquitin E3 ligase inhibitors.

FIG. 19. Kirby-Bauer antibiotic testing. BC-1215 was tested inantibiotic sensitivity tests using Mueller-Hinton agar. Briefly, 6 mmfilter papers containing different amounts of BC-1215 or gentamicin(positive control) were added on the Mueller-Hinton agar pre-exposed toStaphylococcus aureus. The plates were incubated at 37 degrees for 24 h.Zone sizes were measured and marked by a red circle indicating positiveresults. The data suggest that BC-1215 may inhibit bacterial growththrough interaction with the bacterial ApaG protein.

FIGS. 20A-20J is a table depicting benzathine compounds, and assayresults. PBMC cells (0.6 ml at 1.5*10^6/ml) were treated with 2 ug/mlLPS for 16 hrs along with each compound at different concentrations.IL13 and TNFα cytokine release were monitored by ELISA to calculate theIC50. U937 monocyte (0.6 ml at 1.5*10^6/ml) were treated with eachcompound at different concentrations for 16 h. Cells were then stainedwith Trypan blue to differentiate dead cells, and calculate the LD50.Therapeutic index (TI)=LD50/IC50. Compounds marked in red are high valuetargets (low IC50, high LD50) and require further testing in vivo.

FIGS. 21A-21E. BC-1261 reduces P. aeruginosa induced lung inflammation.BC-1261 was administered to mice though an i.p. injection, and mice werethen immediately challenged with P. aeruginosa (strain PA103, 2.5*104cfu/mouse, i.t.) or without (control, CON) for an additional 18 h. Micewere then euthanized and lungs were lavaged with saline, harvested, andthen homogenized. Lavage protein, cell counts and cytokine secretionwere measured in (A-C). D. Lavage cells were processed with cytosin andstained with May-Grunwald and Geimsa. E. H&E staining was performed onlung samples. The data represent n=4 mice/group, *P<0.05 versus PA103

FIGS. 22A-22C. BC-1261 reduces smoke induced chronic lung inflammation.Mice were exposed to cigarette smoke for 5 weeks before one does of i.p.injection of BC1261 (100 ug), 18 h later, mice were euthanized and lungswere lavaged with saline, harvested, and then homogenized. Lavageprotein, cell counts and cytokine secretion were measured in (A-C). Thedata represent n=3 mice/group, *P<0.05 versus con.

FIGS. 23A-23D. BC-1261 reduces TPA induced ear edema. Different dose ofBC-1261 was applied to ears of mice at various doses 30 min after TPAadministration (2 μg/ear). A. Gross comparisons were made with equalvolumes of ethanol as a vehicle control (CON). 18 h after TPAadministration, mice were euthanized and the thickness of the ear wasmeasured using a micrometer (B). Autopsy sample were also taken tomeasure MPO activity (C) and calculate ear edema (D). The data representn=6 mice/group, *P<0.05 versus TPA.

FIGS. 24A-24E. BC-1261 reduces DSS induced acute colonic inflammation.A-D. C57BL6 mice were fed with water ad lib containing 3.5% dextransulfate sodium (DSS) for up to five days. Mice were treated with eithervehicle (control [CON]) or BC-1261 (150 μg) daily (via an i.p.injection), or BC-1261 were administered into drinking water at 30 μg/ml(po). Mice were then euthanized and the length of the colon was measuredand graphed in (A-B). Colonic tissues were also analyzed for TNFα (C)and IL6(D). E. H&E staining was performed on colonic sections. The datarepresent n=4 mice/group, *P<0.05 versus DSS and **P<0.05 versus CON.

FIGS. 25A-25J. BC-1261 reduces DSS induced chronic colonic inflammation.A. C57BL6 mice were fed with water ad lib containing 2% dextran sulfatesodium (DSS) for six days, then switch to water for up to three cycles.BC-1261 was administered into drinking water at 30 μg/ml, starting atday 7. Mice were euthanized at the end of the experiment and the lengthof the colon was measured and graphed in (B-C). Disease index wasmeasured and graphed in (D). Serum cytokine levels were measured in(E-F). Colonic tissues cytokines and MPO activity were also analyzed(GJ). The data represent n=7 mice/group, *P<0.05 versus CON.

FIGS. 26A-26D. Docking study of compound BC-1234 with FBXO3-ApaG domainA. BC-1234 structure. B. BC-1234 interacts with Glu64 and Thr91 residueswithin the FBXO3 ApaG motif. C. The five poses of BC-1234 with the bestdocking scores interacting with FBXO3-ApaG motif. D. BC-1234 werefurther tested in MLE (murine epithelia cells). Briefly, MLE cells weretreated with BC-1234 at different concentrations for 16 h. Cells werethen collected and assayed for Aurora B, cyclin D3, FBXL2 and FBXO3immunoblotting.

FIGS. 27A-27G. BC-1258 Induces G2/M arrest and apoptosis in cancercells. A. PBMCs from 5 controls, AML, and ALL subjects were cultured inRPMI medium for 18 h. Cells were then collected, lysed, and assayed forprotein immunoblotting. B-D. Human leukemia cells (U937, K562 and THP1cells) were treated with BC-1258 at different concentrations for 16 h.Cells were collected and assayed for Aurora B, cyclin D2, cyclin D3 andFBXL2 immunoblotting. E-F. MLE cells were treated with BC-1258 atdifferent concentrations for 16 h, cells were processed by BrdU uptakeand 7-AAD staining followed by FACS cell cycle analysis (E), 2N, 4N, and8N DNA histograms were quantitated and graphed in (F). G. Quantificationof FACS analysis showing levels of apoptotic MLE cells after BC-1258treatment at each time point.

FIGS. 28A-28I. BC-1258 suppresses tumor growth in xenograft. A-E. Effectof BC-1258 and other compounds on growth of U937 tumor implants in nudemice, n=4 mice/group with drug concentration at 30 ug/ml in the drinkingwater. The panel A showed representative images of variable sizes ofxenograft in three nude mice (arrows) after drug treatment. B. Tumorvolume measurements over time (n=4 mice/group, *P<0.05 versus con). D.Tumor tissue from C were weighted and graphed (n=4 mice/group, *P<0.05versus con). E. Tumors from three controls and three drug treated U937implants in mice were collected at the end-point, and assayed forAuroraB, CaM and FBXL2 proteins by immunoblotting. F-I. Serum of eachmice were collected at the end point and processed for creatinine, LDH,ALT and creatine kinase activity.

FIG. 29. ApaG drug binding motif.

FIG. 30. FBXO3-ApaG interaction with compounds BC-1261 and BC-1234.

FIG. 31. FBXO3-ApaG interaction with compound BC-1304.

FIG. 32. FBXO3-ApaG interaction with compound BC-1305.

FIG. 33. FBXO3-ApaG interaction with compound BC-1305 (Secondaryposition).

FIG. 34. FBXO3-ApaG interaction with compound BC-1306.

FIG. 35. FBXO3-ApaG interaction with compound BC-1307.

FIG. 36. FBXO3-ApaG interaction with compound BC-1308.

FIG. 37. FBXO3-ApaG interaction with compound BC-1309.

FIG. 38. Table summarizing toxicity screening for compounds BC-1215 andBC-1261.

SEQUENCE LISTING

The amino acid sequence listed in the accompanying sequence listing isshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822. The Sequence Listing is submitted as an ASCII text file,created on Oct. 10, 2018, 4.47 KB, which is incorporated by referenceherein.

DETAILED DESCRIPTION Terminology

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Also, as usedherein, the term “comprises” means “includes.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

To facilitate review of the various examples of this disclosure, thefollowing explanations of specific terms are provided:

“Acyl” refers to a group having the structure —C(O)R, where R may be,for example, optionally substituted alkyl, optionally substituted aryl,or optionally substituted heteroaryl. “Lower acyl” groups are those thatcontain one to six carbon atoms.

“Acyloxy” refers to a group having the structure —OC(O)R—, where R maybe, for example, optionally substituted alkyl, optionally substitutedaryl, or optionally substituted heteroaryl. “Lower acyloxy” groupscontain one to six carbon atoms.

“Administration” as used herein is inclusive of administration byanother person to the subject or self-administration by the subject.

The term “aliphatic” is defined as including alkyl, alkenyl, alkynyl,halogenated alkyl and cycloalkyl groups. A “lower aliphatic” group is abranched or unbranched aliphatic group having from 1 to 10 carbon atoms.

“Alkanediyl,” “cycloalkanediyl,” “aryldiyl,” “alkanearyldiyl” refers toa divalent radical derived from aliphatic, cycloaliphatic, aryl, andalkanearyl hydrocarbons.

“Alkenyl” refers to a cyclic, branched or straight chain groupcontaining only carbon and hydrogen, and contains one or more doublebonds that may or may not be conjugated.

Alkenyl groups may be unsubstituted or substituted. “Lower alkenyl”groups contain one to six carbon atoms.

The term “alkoxy” refers to a straight, branched or cyclic hydrocarbonconfiguration and combinations thereof, including from 1 to 20 carbonatoms, preferably from 1 to 8 carbon atoms (referred to as a “loweralkoxy”), more preferably from 1 to 4 carbon atoms, that include anoxygen atom at the point of attachment. An example of an “alkoxy group”is represented by the formula —OR, where R can be an alkyl group,optionally substituted with an alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, alkoxy or heterocycloalkyl group.Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy,n-butoxy, i-butoxy, sec-butoxy, tert-butoxy cyclopropoxy, cyclohexyloxy,and the like.

“Alkoxycarbonyl” refers to an alkoxy substituted carbonyl radical,—C(O)OR, wherein R represents an optionally substituted alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl or similar moiety.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A“lower alkyl” group is a saturated branched or unbranched hydrocarbonhaving from 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4carbon atoms. Alkyl groups may be “substituted alkyls” wherein one ormore hydrogen atoms are substituted with a substituent such as halogen,cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. Forexample, a lower alkyl or (C₁-C₆)alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₃-C₆)cycloalkyl(C₁-C₆)alkyl can be cyclopropylmethyl,cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or2-cyclohexylethyl; (C₁-C₆)alkoxy can be methoxy, ethoxy, propoxy,isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, orhexyloxy; (C₂-C₆)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;(C₂-C₆)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₆)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C₁-C₆)alkylcan be iodomethyl, bromomethyl, chloromethyl, fluoromethyl,trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, orpentafluoroethyl; hydroxy(C₁-C₆)alkyl can be hydroxymethyl,1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl,3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl,5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl;(C₁-C₆)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₁-C₆)alkylthio can be methylthio, ethylthio,propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, orhexylthio; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy,isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

“Alkynyl” refers to a cyclic, branched or straight chain groupcontaining only carbon and hydrogen, and contains one or more triplebonds. Alkynyl groups may be unsubstituted or substituted. “Loweralkynyl” groups are those that contain one to six carbon atoms.

The term “amine” or “amino” refers to a group of the formula —NRR′,where R and R′ can be, independently, hydrogen or an alkyl, alkenyl,alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group. For example, an “alkylamino” or “alkylatedamino” refers to —NRR′, wherein at least one of R or R′ is an alkyl.

“Aminocarbonyl” alone or in combination, means an amino substitutedcarbonyl (carbamoyl) radical, wherein the amino radical may optionallybe mono- or di-substituted, such as with alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyland the like. An aminocarbonyl group may be —N(R)—C(O)—R (wherein R is asubstituted group or H). A suitable aminocarbonyl group is acetamido.

The term “amide” or “amido” is represented by the formula —C(O)NRR′,where R and R′ independently can be a hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above.

An “analog” is a molecule that differs in chemical structure from aparent compound, for example a homolog (differing by an increment in thechemical structure or mass, such as a difference in the length of analkyl chain or the inclusion of one of more isotopes), a molecularfragment, a structure that differs by one or more functional groups, ora change in ionization. An analog is not necessarily synthesized fromthe parent compound. A derivative is a molecule derived from the basestructure.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats. The term subject appliesregardless of the stage in the organism's life-cycle. Thus, the termsubject applies to an organism in utero or in ovo, depending on theorganism (that is, whether the organism is a mammal or a bird, such as adomesticated or wild fowl).

“Aryl” refers to a monovalent unsaturated aromatic carbocyclic grouphaving a single ring (e.g., phenyl) or multiple condensed rings (e.g.,naphthyl or anthryl), which can optionally be unsubstituted orsubstituted. A “heteroaryl group,” is defined as an aromatic group thathas at least one heteroatom incorporated within the ring of the aromaticgroup. Examples of heteroatoms include, but are not limited to,nitrogen, oxygen, sulfur, and phosphorous. Heteroaryl includes, but isnot limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzooxazolyl, quinoxalinyl, and the like. The aryl or heteroaryl groupcan be substituted with one or more groups including, but not limitedto, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone,aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl or heteroarylgroup can be unsubstituted.

The term “aralkyl” refers to an alkyl group wherein an aryl group issubstituted for a hydrogen of the alkyl group. An example of an aralkylgroup is a benzyl group.

“Aryloxy” or “heteroaryloxy” refers to a group of the formula —OAr,wherein Ar is an aryl group or a heteroaryl group, respectively.

Atomic coordinates or structure coordinates refers to mathematicalcoordinates derived from mathematical equations related to the patternsobtained on diffraction of a monochromatic beam of X-rays by the atoms(scattering centers) such as a protein. In some examples that proteincan be FBXO3 protein in a crystal. The diffraction data are used tocalculate an electron density map of the repeating unit of the crystal.The electron density maps are used to establish the positions of theindividual atoms within the unit cell of the crystal. In one example,the term “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays, such as by the atoms of aFBXO3 protein in crystal form. Those of ordinary skill in the artunderstand that a set of structure coordinates determined by X-raycrystallography is not without standard error. For the purpose of thisdisclosure, any set of structure coordinates that have a root meansquare deviation of protein backbone atoms (N, Cα, C and 0) of less thanabout 1.0 Angstroms when superimposed, such as about 0.75, or about 0.5,or about 0.25 Angstroms, using backbone atoms, shall (in the absence ofan explicit statement to the contrary) be considered identical.

The term “carboxylate” or “carboxyl” refers to the group —COO⁻ or —COOH.

The term “co-administration” or “co-administering” refers toadministration of a FBXO3 inhibitor with at least one other therapeuticagent within the same general time period, and does not requireadministration at the same exact moment in time (althoughco-administration is inclusive of administering at the same exact momentin time). Thus, co-administration may be on the same day or on differentdays, or in the same week or in different weeks. The additionaltherapeutic agent may be included in the same composition as the FBXO3inhibitor.

The term “cycloalkyl” refers to a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and the like. The term “heterocycloalkyl group” is acycloalkyl group as defined above where at least one of the carbon atomsof the ring is substituted with a heteroatom such as, but not limitedto, nitrogen, oxygen, sulfur, or phosphorous.

The term “ester” refers to a carboxyl group having the hydrogen replacedwith, for example, a C₁₋₆alkyl group (“carboxylC₁₋₆alkyl” or“alkylester”), an aryl or aralkyl group (“arylester” or “aralkylester”)and so on. CO₂C₁₋₃alkyl groups are preferred, such as for example,methylester (CO₂Me), ethylester (CO₂Et) and propylester (CO₂Pr) andincludes reverse esters thereof (e.g. —OCOMe, —OCOEt and —OCOPr).

The term “halogen” refers to fluoro, bromo, chloro and iodosubstituents.

The terms “halogenated alkyl” or “haloalkyl group” refer to an alkylgroup as defined above with one or more hydrogen atoms present on thesegroups substituted with a halogen (F, Cl, Br, I).

The term “hydroxyl” is represented by the formula —OH.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

“N-heterocyclic” or “N-heterocycle” refers to mono or bicyclic rings orring systems that include at least one nitrogen heteroatom. The rings orring systems generally include 1 to 9 carbon atoms in addition to theheteroatom(s) and may be saturated, unsaturated or aromatic (includingpseudoaromatic). The term “pseudoaromatic” refers to a ring system whichis not strictly aromatic, but which is stabilized by means ofdelocalization of electrons and behaves in a similar manner to aromaticrings. Aromatic includes pseudoaromatic ring systems, such as pyrrolylrings.

Examples of 5-membered monocyclic N-heterocycles include pyrrolyl,H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including1,2,3 and 1,2,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl,isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls),tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls), anddithiazolyl. Examples of 6-membered monocyclic N-heterocycles includepyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, and triazinyl. The heterocycles may beoptionally substituted with a broad range of substituents, andpreferably with C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl,halo, hydroxy, mercapto, trifluoromethyl, amino, cyano or mono ordi(C₁₋₆alkyl)amino. The N-heterocyclic group may be fused to acarbocyclic ring such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl,and anthracenyl.

Examples of 8, 9 and 10-membered bicyclic heterocycles include 1Hthieno[2,3-c]pyrazolyl, indolyl, isoindolyl, benzoxazolyl,benzothiazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolyl,indazolyl, isoquinolinyl, quinolinyl, quinoxalinyl, purinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, benzotriazinyl, and the like.These heterocycles may be optionally substituted, for example with C₁₋₆alkyl, C₁₋₆ alkoxy, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, hydroxy, mercapto,trifluoromethyl, amino, cyano or mono or di(C₁₋₆alkyl)amino. Unlessotherwise defined optionally substituted N-heterocyclics includespyridinium salts and the N-oxide form of suitable ring nitrogens.

“Nitro” refers to an R-group having the structure —NO₂.

An “R-group” or “substituent” refers to a single atom (for example, ahalogen atom) or a group of two or more atoms that are covalently bondedto each other, which are covalently bonded to an atom or atoms in amolecule to satisfy the valency requirements of the atom or atoms of themolecule, typically in place of a hydrogen atom. Examples ofR-groups/substituents include alkyl groups, hydroxyl groups, alkoxygroups, acyloxy groups, mercapto groups, and aryl groups.

The term “subject” includes both human and non-human subjects, includingbirds and non-human mammals, such as non-human primates, companionanimals (such as dogs and cats), livestock (such as pigs, sheep, cows),as well as non-domesticated animals, such as the big cats. The termsubject applies regardless of the stage in the organism's life-cycle.Thus, the term subject applies to an organism in utero or in ovo,depending on the organism (that is, whether the organism is a mammal ora bird, such as a domesticated or wild fowl).

“Substituted” or “substitution” refer to replacement of a hydrogen atomof a molecule or an R-group with one or more additional R-groups. Unlessotherwise defined, the term “optionally-substituted” or “optionalsubstituent” as used herein refers to a group which may or may not befurther substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or3, more preferably 1 or 2 groups. The substituents may be selected, forexample, from C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₃₋₈cycloalkyl,hydroxyl, oxo, C₁₋₆alkoxy, aryloxy, C₁₋₆alkoxyaryl, halo, C₁₋₆alkylhalo(such as CF₃ and CHF₂), C₁₋₆alkoxyhalo (such as OCF₃ and OCHF₂),carboxyl, esters, cyano, nitro, amino, substituted amino, disubstitutedamino, acyl, ketones, amides, aminoacyl, substituted amides,disubstituted amides, thiol, alkylthio, thioxo, sulfates, sulfonates,sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl,sulfonylamides, substituted sulfonamides, disubstituted sulfonamides,aryl, arC₁₋₆ alkyl, heterocyclyl and heteroaryl wherein each alkyl,alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groupscontaining them may be further optionally substituted. Optionalsubstituents in the case N-heterocycles may also include but are notlimited to C₁₋₆alkyl i.e. N—C₁₋₃alkyl, more preferably methylparticularly N-methyl.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. For example, a therapeutically amount may be an amountof a FBXO3 inhibitor that is sufficient to inhibit inflammation in asubject. Ideally, a therapeutically effective amount of an agent is anamount sufficient to inhibit or treat the disease or condition withoutcausing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” refers to inhibitingthe full development of a disease, for example, in a subject who is atrisk for a disease such as cancer. “Preventing” a disease or conditionrefers to prophylactic administering a composition to a subject who doesnot exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing a pathology or condition,or diminishing the severity of a pathology or condition. In certainembodiments disclosed herein, the treatment inhibits inflammation in asubject.

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

Some of the compounds described herein may also exist in theirtautomeric form.

Prodrugs of the disclosed compounds also are contemplated herein. Aprodrug is an active or inactive compound that is modified chemicallythrough in vivo physiological action, such as hydrolysis, metabolism andthe like, into an active compound following administration of theprodrug to a subject. The term “prodrug” as used throughout this textmeans the pharmacologically acceptable derivatives such as esters,amides and phosphates, such that the resulting in vivo biotransformationproduct of the derivative is the active drug as defined in the compoundsdescribed herein. Prodrugs preferably have excellent aqueous solubility,increased bioavailability and are readily metabolized into the activeinhibitors in vivo. Prodrugs of a compounds described herein may beprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either by routine manipulationor in vivo, to the parent compound. The suitability and techniquesinvolved in making and using prodrugs are well known by those skilled inthe art. F or a general discussion of prodrugs involving esters seeSvensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard,Design of Prodrugs, Elsevier (1985).

The term “prodrug” also is intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when the prodrug is administered to a subject. Since prodrugs oftenhave enhanced properties relative to the active agent pharmaceutical,such as, solubility and bioavailability, the compounds disclosed hereincan be delivered in prodrug form. Thus, also contemplated are prodrugsof the presently disclosed compounds, methods of delivering prodrugs andcompositions containing such prodrugs. Prodrugs of the disclosedcompounds typically are prepared by modifying one or more functionalgroups present in the compound in such a way that the modifications arecleaved, either in routine manipulation or in vivo, to yield the parentcompound. Prodrugs include compounds having a phosphonate and/or aminogroup functionalized with any group that is cleaved in vivo to yield thecorresponding amino and/or phosphonate group, respectively. Examples ofprodrugs include, without limitation, compounds having an acylated aminogroup and/or a phosphonate ester or phosphonate amide group. Inparticular examples, a prodrug is a lower alkyl phosphonate ester, suchas an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated.A variety of suitable protecting groups for use with the disclosedcompounds are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions that will notaffect the remaining portion of the molecule. These methods are wellknown in the art and include acid hydrolysis, hydrogenolysis and thelike. One preferred method involves the removal of an ester, such ascleavage of a phosphonate ester using Lewis acidic conditions, such asin TMS-Br mediated ester cleavage to yield the free phosphonate. Asecond preferred method involves removal of a protecting group, such asremoval of a benzyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxy-based group, including t-butoxycarbonyl protecting groups can be removed utilizing an inorganic ororganic acid, such as HCl or trifluoroacetic acid, in a suitable solventsystem, such as water, dioxane and/or methylene chloride. Anotherexemplary protecting group, suitable for protecting amino and hydroxyfunctions amino is trityl. Other conventional protecting groups areknown and suitable protecting groups can be selected by those of skillin the art in consultation with Greene and Wuts, Protective Groups inOrganic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When anamine is deprotected, the resulting salt can readily be neutralized toyield the free amine. Similarly, when an acid moiety, such as aphosphonic acid moiety is unveiled, the compound may be isolated as theacid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one ormore asymmetric centers; thus these compounds can exist in differentstereoisomeric forms. Accordingly, compounds and compositions may beprovided as individual pure enantiomers or as stereoisomeric mixtures,including racemic mixtures. In certain embodiments the compoundsdisclosed herein are synthesized in or are purified to be insubstantially enantiopure form, such as in a 90% enantiomeric excess, a95% enantiomeric excess, a 97% enantiomeric excess or even in greaterthan a 99% enantiomeric excess, such as in enantiopure form.

Groups which are substituted (e.g. substituted alkyl), may in someembodiments be substituted with a group which is substituted (e.g.substituted aryl). In some embodiments, the number of substituted groupslinked together is limited to two (e.g. substituted alkyl is substitutedwith substituted aryl, wherein the substituent present on the aryl isnot further substituted). In some embodiments, a substituted group isnot substituted with another substituted group (e.g. substituted alkylis substituted with unsubstituted aryl).

Overview

It has been discovered that pathogens activate a relativelyrecently-identified ubiquitin E3 ligase subunit, termed FBXO3 (SEQ ID1),which is sufficient to ubiquitinate and mediate proteasomal degradationof another relatively recently-identified ubiquitin E3 ligase subunit,termed FBXL2. Further, it has also been discovered that FBXL2 acts as a“break” on inflammation, by targeting the TRAF family of proteins fortheir disposal in epithelia and monocytes. Thus, pathogens, viaactivation of FBXO3, result in FBXL2 ubiquitination and degradationresulting in increased immunoreactive TRAFs, increased cytokineproduction, and impaired lung stability. Specifically, the datadisclosed herein show that i) FBXL2 targets six TRAF family proteins(TRAF1-6) for their ubiquitination and degradation, (ii) FBXO3specifically targets FBXL2 for its ubiquitination and degradation, (iii)glycogen synthase kinase (GSK3β) phosphorylates FBXL2 thereby serving asa novel molecular signal for FBXO3 ubiquitination of FBXL2, and (iv)compared to wild-type (Wt) FBXO3, expression of a naturally occurringFBXO3 point mutant (FBXO3V220I) fails to stimulate cytokine productionafter P. aeruginosa infection, and expression of FBXO3V220I lessens theseverity of inflammatory lung injury in murine models of pneumonia.

The discovery of FBXO3 is of particular importance as it contains abacterial-like molecular signature within its tertiary structure notdetected in mammalian proteins. This motif, termed Apa G, led to thepresently disclosed development of a highly unique, selective phylum ofsmall molecule therapeutics that block FBXO3 activity, reduce TRAFlevels to native levels, profoundly inhibit cytokine release from humancells, and lessen the severity of inflammation in septic animal models.A series of small molecule inhibitors of FBXO3 were generated that whentested attenuate lipopolysaccharide (LPS)-induced cytokine secretionfrom human peripheral blood mononuclear cells. In one embodiment, theFBXO3 inhibitor BC-1215 inhibits inflammation and prevents tissue damagein several animal models.

Provided herein is a new molecular model of innate immunity as itrelates to cytokine signaling. Two previously poorly characterizedproteins (FBXO3, FBXL2) newly linked to the cytokine response throughTRAF protein signaling have been uncovered. The studies disclosed hereinare the first to elucidate the enzymatic behavior FBXO3 that appears toactivate the FBXL2-TRAF-cytokine axis. Based on the previouslyunrecognized novel mechanism of FBXO3 activity in the TRAF inflammatorypathway, the agents disclosed herein target a unique prokaryoticmolecular signature within the F box protein. Disclosed herein arebenzathine compounds that serve as highly selective small moleculeinhibitors of FBXO3, and that may be useful in the prophylaxis andtreatment of septic shock, pneumonia, and other inflammatory conditions.

Compounds

Disclosed herein in one embodiment are FBXO3 inhibitors. IllustrativeFBXO3 inhibitors include benzathine compounds, optionally-substituteddiaminoalkanes (e.g., 1,10-diaminodecane), substituted quinolines (e.g.,quinidine, hydroxychloroquine, primaquine), haematoxylin,tetramethylenebis, naphthacaine, ampicillin, and elliptine, andpharmaceutically acceptable salts and esters thereof.

The benzathine compound may be benzathine or a benzathine analog. Incertain embodiments the benzathine compound is not benzathinepenicillin. In certain embodiments the benzathine analog includes adivalent diamine core moiety, a first aryl-containing moiety at a firstterminal end of the divalent diamine core moiety, and a secondaryl-containing moiety at a second terminal end of the divalent diaminecore moiety. Each amino groups of the diamine group may be individually—NH— or —NR—, wherein R is a substituted group as described such as alower alkyl, alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl, aryl,carboxyl, or ester. The divalent diamine core moiety may include anoptionally-substituted alkanediyl, an optionally-substitutedcycloalkanediyl, an optionally-substituted aryldiyl, or anoptionally-substituted alkanearyldiyl positioned between the two aminogroups. In certain embodiments the two amino groups of the diamine maytogether with carbon atoms form a heteroaryldiyl group. The terminalaryl-containing groups may each individually be an aralkyl group(preferably a benzyl group) or an N-heteroaralkyl group such as-alkyl-pyrazinyl, -alkyl-pyrimidinyl, -alkyl-pyridazinyl, or-alkyl-pyridinyl. The aryl ring of the aralkyl group may be substitutedwith an optionally-substituted N-heterocyclic group. In certainembodiments, the optionally-substituted N-heterocyclic group is locatedat a ring position para to the point of attachment of the aralkyl groupto the divalent diamine core moiety.

Illustrative benzathine analogs include optionally-substitutedN-heterocyclic-substituted benzathines. In certain embodiments, thebenzathine analogs include two phenyl rings, wherein at least one, andpreferably both, of the phenyl rings are substituted with anoptionally-substituted N-heterocyclic group, whichoptionally-substituted N-heterocyclic may be the same or different. Incertain embodiments, the optionally-substituted N-heterocyclic group islocated at a ring position para to the point of attachment of the phenylring to the benzathine molecular scaffold.

Illustrative N-heterocyclic groups include, for example, pyrrolyl,H-pyrrolyl, pyrrolinyl, pyrrolidinyl, oxazolyl, oxadiazolyl, (including1,2,3; 1,2,4; and 1,3,4 oxadiazolyls) isoxazolyl, furazanyl, thiazolyl,isothiazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, triazolyl (including 1,2,3 and 1,3,4 triazolyls),tetrazolyl, thiadiazolyl (including 1,2,3 and 1,3,4 thiadiazolyls),dithiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl,morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl. Particularlypreferred N-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl,oxadiazolyl and pyrimidinyl.

The benzathine analogs, or pharmaceutically acceptable salts or estersthereof, may have structure of formula I:

wherein X is a divalent or tetravalent linking moiety; and

R¹-R¹⁰ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy.

In certain embodiments of formula I, X is an optionally-substitutedalkanediyl, an optionally-substituted cycloalkanediyl, anoptionally-substituted aryldiyl, or an optionally-substitutedalkanearyldiyl. For example, X may be an alkanediyl having a structureof —C_(n)H_(2n)—wherein n is 1 to 10, more preferably 2 to 5; X may be a—C₆H₁₀-cycloalkanediyl; or X may be a —C₆H4— aryldiyl. A particularlypreferred X moiety is —CH₂—CH₂—.

In certain embodiments of formula I, X is a tetravalent moiety that isderived from a spiro structure wherein the nitrogen atoms of the diaminecore form N-heteroatoms of the spiro structure. For example, X togetherwith the diamine may form a diazaspirodecane. An example of adiazaspirodecane is shown below in formula VI.

In certain embodiments of formula I, at least one of R¹-R¹⁰ is not H. Incertain embodiments of formula I, at least one of R³ or R⁸ is anoptionally-substituted alkyl, optionally-substituted alkoxy,optionally-substituted aryl, optionally-substituted cycloalkyl,optionally-substituted heterocyclic, halogen, amino, or hydroxy. Incertain embodiments of formula I, at least one of R³ or R⁸, andpreferably both of R³ and R⁸, is an unsubstituted alkoxy,aryl-substituted alkoxy, halo-substituted alkoxy, aryl,optionally-substituted heterocyclic, halogen, amino, or hydroxy. Incertain embodiments of formula I, at least one of R¹-R¹⁰ is anN-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic.In certain embodiments of formula I, at least one of R³ or R⁸, andpreferably both of R³ and R⁸, is an N-heterocyclic, particularly a5-membered or 6-membered N-heterocyclic. Illustrative N-heterocyclicgroups include, for example, pyrrolyl, H-pyrrolyl, pyrrolinyl,pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3; 1,2,4; and 1,3,4oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl(including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl(including 1,2,3 and 1,3,4 thiadiazolyls), dithiazolyl, pyridyl,pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, and triazinyl. Particularly preferredN-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl, andpyrimidinyl. Especially preferred N-heterocyclic groups includeimidazolyl, pyridyl, and pyrazolyl. In certain embodiments of formula I,R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are each H. In certain embodiments offormula I, R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are each H; X is anoptionally-substituted alkanediyl, and R³ and R⁸ are each individuallyan optionally-substituted 5-membered or 6-membered N-heterocyclic. Incertain embodiments of formula I, R³ and R⁸ are each the same group.

Disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula II:

wherein X is a divalent linking moiety; and

R¹-R¹⁰ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy, provided that at least one of R³ or R⁸ is anoptionally-substituted alkyl, a substituted alkoxy,optionally-substituted aryl, optionally-substituted cycloalkyl,optionally-substituted heterocyclic, or halogen.

In certain embodiments of formula II, X is an optionally-substitutedalkanediyl, an optionally-substituted cycloalkanediyl, anoptionally-substituted aryldiyl, or an optionally-substitutedalkanearyldiyl. For example, X may be an alkanediyl having a structureof —C_(n)H_(2n)—wherein n is 1 to 10, more preferably 2 to 5; X may be a—C₆H₁₀-cycloalkanediyl; or X may be a —C₆H₄— aryldiyl. A particularlypreferred X moiety is —CH₂—CH₂—.

In certain embodiments of formula II, at least one of R¹-R¹⁰ is anN-heterocyclic, particularly a 5-membered or 6-membered N-heterocyclic.In certain embodiments of formula II, at least one of R³ or R⁸, andpreferably both of R³ and R⁸, is an N-heterocyclic, particularly a5-membered or 6-membered N-heterocyclic. Illustrative N-heterocyclicgroups include, for example, pyrrolyl, H-pyrrolyl, pyrrolinyl,pyrrolidinyl, oxazolyl, oxadiazolyl, (including 1,2,3; 1,2,4; and 1,3,4oxadiazolyls) isoxazolyl, furazanyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, triazolyl(including 1,2,3 and 1,3,4 triazolyls), tetrazolyl, thiadiazolyl(including 1,2,3 and 1,3,4 thiadiazolyls), dithiazolyl, pyridyl,pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, and triazinyl. Particularly preferredN-heterocyclic groups include imidazolyl, pyridyl, pyrazolyl,oxadiazolyl and pyrimidinyl. Especially preferred N-heterocyclic groupsinclude imidazolyl, pyridyl, and pyrazolyl. In certain embodiments offormula II, R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are each H. In certainembodiments of formula II, R¹, R², R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are eachH; X is an optionally-substituted alkanediyl, and R³ and R⁸ are eachindividually an optionally-substituted 5-membered or 6-memberedN-heterocyclic. In certain embodiments of formula II, R³ and R⁸ are eachthe same group.

Disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula III:

wherein X is a divalent linking moiety; and

R²-R⁵ and R⁷-R¹⁰ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy.

In certain embodiments of formula III, X is an optionally-substitutedalkanediyl, an optionally-substituted cycloalkanediyl, anoptionally-substituted aryldiyl, or an optionally-substitutedalkanearyldiyl. For example, X may be an alkanediyl having a structureof —C_(n)H_(2n)—wherein n is 1 to 10, more preferably 2 to 5; X may be a—C₆H₁₀— cycloalkanediyl; or X may be a —C₆H₄— aryldiyl. A particularlypreferred X moiety is —CH₂—CH₂—. In certain embodiments of formula III,R²-R⁵ and R⁷-R¹⁰ are each individually H.

Disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula IV:

wherein X is a divalent linking moiety; and

R²-R⁴ and R⁷-R⁹ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy.

In certain embodiments of formula IV, X is an optionally-substitutedalkanediyl, an optionally-substituted cycloalkanediyl, anoptionally-substituted aryldiyl, or an optionally-substitutedalkanearyldiyl. For example, X may be an alkanediyl having a structureof —C_(n)H_(2n)—wherein n is 1 to 10, more preferably 2 to 5; X may be a—C₆H₁₀— cycloalkanediyl; or X may be a —C₆H₄— aryldiyl. A particularlypreferred X moiety is —CH₂—CH₂—. In certain embodiments of formula IV,R²-R⁵ and R⁷-R¹⁰ are each individually H.

Also disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula V:

wherein X is a divalent linking moiety as described above;

R²⁰ and R²¹ are each individually selected from hydrogen, lower alkyl,alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl, aryl, carboxyl, orester; and

R²² and R²³ are each individually selected from anoptionally-substituted aryl or an optionally-substituted N-heterocycle,provided that at least one of R²² or R²³ is an optionally-substitutedN-heterocycle.

In certain preferred embodiments of formula V, R²³ is an N-heterocycleand R²² is an N-heterocycle-substituted phenyl, particularly apara-substituted N-heterocycle-phenyl.

Also disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula VI:

wherein Ar¹ and Ar² are each independently optionally-substituted arylor optionally-substituted N-heterocyclic. Illustrative N-heterocyclicgroups include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl,piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, and triazinyl.The aryl (particularly phenyl) or N-heterocyclic (particularlypyrimidinyl) may be substituted with alkyl (particularly lower alkyl),alkoxy (particularly methoxy), aminocarbonyl (particularly acetamido),halogen, or alkyl-substituted thiol (particularly —S—CH₂CH₃).

Also disclosed herein in a further embodiment are compounds, orpharmaceutically acceptable salts or esters thereof, having a structureof formula VII:

wherein X is a divalent or tetravalent linking moiety;

R³¹-R³⁵ are each individually H, optionally-substituted alkyl,optionally-substituted alkoxy, optionally-substituted aryl,optionally-substituted cycloalkyl, optionally-substituted heterocyclic,halogen, amino, or hydroxy; and

R³⁶ is hydrogen, optionally-substituted lower alkyl,optionally-substituted alkoxy, hydroxy, acyl, acyloxy, alkoxycarbonyl,optionally-substituted aryl, carboxyl, or optionally-substituted ester.

In certain embodiments of formula VII, X is an optionally-substitutedalkanediyl, an optionally-substituted cycloalkanediyl, anoptionally-substituted aryldiyl, or an optionally-substitutedalkanearyldiyl. For example, X may be an alkanediyl having a structureof —C_(n)H_(2n)—wherein n is 1 to 10, more preferably 2 to 5; X may be a—C₆H₁₀— cycloalkanediyl; or X may be a —C₆H₄— aryldiyl. A particularlypreferred X moiety is —CH₂—CH₂—.

In certain embodiments of formula VII, X is a tetravalent moiety that isderived from a spiro structure wherein the nitrogen atoms of the diaminecore form N-heteroatoms of the spiro structure. For example, X togetherwith the diamine may form a diazaspirodecane. An example of adiazaspirodecane is shown above in formula VI.

In certain embodiments of formula VII, at least one of R³¹-R³⁵ is not H.In certain embodiments of formula VII, at least one of R³ or R⁸ is anoptionally-substituted alkyl, optionally-substituted alkoxy,optionally-substituted aryl, optionally-substituted cycloalkyl,optionally-substituted heterocyclic, halogen, amino, or hydroxy. Incertain embodiments of formula I, ^(R34) is an unsubstituted alkoxy,aryl-substituted alkoxy, halo-substituted alkoxy, aryl,optionally-substituted heterocyclic, halogen, amino, or hydroxy.

In certain embodiments of formula VII, R³⁶ is hydrogen, lower alkyl(particularly methyl, ethyl, or butyl), methoxy, hydroxy, —C(O)R⁴⁰,where R⁴⁰ is a lower alkyl, —OC(O)R⁴¹—, where R⁴¹ is a lower alkyl,—C(O)OR⁴², wherein R⁴² is a lower alkyl, phenyl, or —COOH.

In certain embodiments of formulae I-VII, the compounds may be in theform of a salt. For example, the diamine moiety within the benzathinecompound structure may form a salt with an anion such as acetate (e.g.,compound BC-1215 HAc), carbonate, halide, citrate, nitrate, nitrite,phosphate, phosphonate, sulfate, sulfonate, or lactic acid. In certainembodiments the compounds of formulae I-VII are water soluble thusenabling their salt formation. The water solubility of the compoundsalso enables formulation of the compounds into aerosol delivery for thelungs, oral administration, or emulsions for topical administration.

Illustrative compounds of formulae I and II are shown in table 1 of FIG.20.

Illustrative compounds are also shown below:

Methods of Use

In one embodiment the compounds disclosed herein may be used fortreating inflammatory disorders, particularly inflammatory disordersthat are mediated by cytokine release, especially a cytokine storm. Forexample, the compounds disclosed herein may be used for treatinginflammatory disorders that underlie numerous human diseasescharacterized by a highly activated immune system that leads tosecretion of large amounts of circulating pro-inflammatory cytokinesafter infection with virulent pathogens, in response to host cellinjury, or related irritants that activate receptors on immune effectorcells (T-cells, macrophages, etc.). A central feature of theseinfectious disorders is the burst in cytokine release, i.e. cytokinestorm, from pro-inflammatory cells including macrophages, lymphocytes,and PMNs. Under many conditions, the cytokine storm is exaggerated(hypercytokinemia) and results in a fatal immune reaction with constantactivation of immune effector cells that produce sustained andsupraphysiologic levels of TNFα, IL-β, and IL-6 that leads to profoundtissue injury. The compounds disclosed herein may inhibit the release ofpro-inflammatory cytokines (e.g., TNFα, IL-β, and/or IL-6). In certainembodiments, the compounds disclosed herein are panreactive to numerousinjurious cytokines. The compounds disclosed herein inhibit inflammationand prevent tissue damage (e.g., lung damage, particularly lung damagefrom bacterial infection) in a subject. For example, the compoundsdisclosed herein may inhibit hypercytokinemia, and/or may prevent ordiminish supraphysiologic levels of TNFα, IL-β, and/or IL-6 or relatedinjurious molecules.

Inflammatory disorders that may be treated by the compounds disclosedherein include any disorder possessing an inflammatory component.Illustrative inflammatory disorders include acute and chronicinflammation disorders such as asthma, chronic obstructive lung disease,pulmonary fibrosis, pneumonitis (including hypersensitivity pneumonitisand radiation pneumonitis), pneumonia, cystic fibrosis, psoriasis,arthritis/rheumatoid arthritis, rhinitis, pharyngitis, cystitis,prostatitis, dermatitis, allergy including hayfever, nephritis,conjunctivitis, encephalitis, meningitis, opthalmitis, uveitis,pleuritis, pericarditis, myocarditis, atherosclerosis, humanimmunodeficiency virus related inflammation, diabetes, osteoarthritis,psoriatic arthritis, inflammatory bowel disease (Crohn's disease,ulcerative colitis)/colitis, sepsis, vasculitis, bursitis, connectivetissue disease, autoimmune diseases such as systemic lupus erythematosis(SLE), polymyalgia rheumatica, scleroderma, Wegener's granulomatosis,temporal arteritis, vasculitis, cryoglobulinemia, and multiplesclerosis, viral or influenza-induced inflammation, or edema. Thecompounds disclosed herein may be particularly effective for treatingsepsis, pneumonia, influenza-induced inflammation, edema, neuropathy,colitis, arthritis, Crohn's disease, diabetes, skin, eye and earinflammation (e.g., psoriasis, uveitis/opthalmitis, external otitis),systemic lupus erythematosis (SLE), and systemic lupus erythematosis(SLE). The compounds disclosed herein may be useful for treatinginflammation and tissue damage induced by pathogenic infection with, forexample, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcuspneumoniae, Haemophilus influenza, or Escherichia coli. The compoundsdisclosed herein may be especially effective for treating sepsis orpneumonia.

In certain embodiments the compounds disclosed herein may beantibacterial agents. The compounds may inhibit bacterial growth(function as a bacteriostatic) of, for example, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenza,or Escherichia coli. The compounds may inhibit bacterial growth throughinteraction with the bacterial ApaG protein. The bacterial growth may beinhibited in a subject by administering the compound to the subject.Bacterial growth on a surface of an object (e.g., a food item, asurgical implement, a kitchen surface, a hospital surface, etc.) may beinhibited by administering or applying the compound to the surface ofthe object.

In certain embodiments the compounds disclosed herein may be used fortreating other FBXO3-mediated disorders or injuries such as, forexample, malaria, toxic lung exposure, cancer, Alzheimer's, or aburn-related injury. Illustrative cancers include leukemia, lymphoma,bronchogenic carcinoma, adenocarcinoma of the breast, colon, ovary,thyroid, pancreas, stomach, and prostate, squamous cell cancer, smallcell cancer, melanoma, sarcoma, and metastatic cancer. Since an FBXO3inhibitor up-regulates FBXL2, other substrates of FBXL2 such as cyclinD2/3, Aurora B protein will be degraded upon FBXO3 inhibitor treatment.Since Cyclin D2/3 and Aurora B are well-described oncoproteins, thusFBXO3 inhibitor may inhibit cancer proliferation through inhibitingcyclins and Aurora B protein.

Another embodiment disclosed herein is a method for inhibitingpro-inflammatory cytokine release in a subject, comprising administeringto the subject an FBXO3 inhibitor. The FBXO3 inhibitor inhibits FBXO3activity, reduces TRAF protein levels in cells, inhibits cytokinerelease from cells, and lessens the severity of inflammation in a septicsubject. In certain embodiments, the FBXO3 inhibitor reduces theconcentration of TRAF proteins (e.g., TRAF2, TRAF5 and TRAF6) in cellsin a subject that has been subjected to a cytokine-inducing event suchas an infection. By targeting TRAF-mediated cytokine release, an FBXO3inhibitor may avoid the severe long-term effects of corticosteroids thatsuppress inflammation at multiple biological pathways, but provide abroader systemic effect relative to anti-inflammatories targeted to asingle cytokine. In certain embodiments analysis of inflammatory bloodcells in subjects treated with FBXO3 inhibitors will show reduced TRAFprotein levels.

In certain embodiments, the compounds disclosed herein target a“bacterial-like” molecular signature (the ApaG domain (SEQ ID1, residues278-400)) identified within FBXO3 that is not identified in otherproteins within mammalian host cells. This feature is highly attractiveas it potentially confers drug selectivity with limited off-targeteffects. In particular, an FBXO3 inhibitor, such as the compoundsdisclosed herein, occupies an ApaG domain cavity of the FBXO3 protein.

An FBXO3-ApaG motif 3D structure was generated from homology model basedon crystal structure of ApaG protein (2F1E.pdb) from Xanthomonasaxonopodis pv. Citri. In certain embodiments, an FBXO3 inhibitorcontacts and interacts with amino acid residues Y308, N335, E341, T368and S370 that are located in the ApaG domain cavity. For example, anFBXO3 inhibitor may couple with the amino acid residues via hydrogenbonding, Van der Waals forces, salt-bridge formation, or covalentbonding. In certain embodiments, an FBXO3 inhibitor includes at leastone amine group that forms a salt-bridge within 4 angstroms fromglutamic acid 341 carboxyl group, and at least one nitrogen- oroxygen-containing group that forms a hydrogen bond within 3 angstromsfrom threonine 368 hydroxyl group, serine 370 hydroxyl group, asparagine335 carboxamide group, and tyrosine 308 hydroxyl group.

In certain embodiments, the subject is in need of, or has beenrecognized as being in need of, treatment with an FBXO3 inhibitor. Thesubject may be selected as being amenable to treatment with an FBXO3inhibitor. For example, the subject may be in need of ananti-inflammatory agent that inhibits inflammation caused by at leasttwo different pro-inflammatory cytokines.

Currently, synthetic glucocorticoids are used in the treatment of a widerange of inflammatory disorders; its primary anti-inflammatory mechanisminvolves blocking lipocortin 1 synthesis, followed by suppressingphospholipase A2 action and modulating levels of two classes ofpro-inflammatory products such as prostaglandins and leukotrienes.However, glucocorticoids have many other target proteins in vivo; thus,its non-specificity with off-target effects may cause a variety ofadverse effects such as hyperglycemia, insulin resistance, diabetesmellitus, osteoporosis, cataracts, anxiety, depression, colitis,hypertension, ictus, erectile dysfunction, hypogonadism, hypothyroidism,amenorrhea, and retinopathy. Based on the novel, selective, mechanism ofFBXO3 inhibitors, the compounds disclosed herein may provide bettertoxicity profile with potent in vivo activity.

The compounds disclosed herein regulate inflammation through arelatively new E3 ligase subunit, FBXO3, and its downstream target,TRAFs proteins. Thus, it represents a totally distinct mechanism ofaction from glucocorticoids and existing anti-inflammatories such asnonsteroidal anti-inflammatory agents (NSAIDs).

Pharmaceutical Compositions

Another aspect of the disclosure includes pharmaceutical compositionsprepared for administration to a subject and which include atherapeutically effective amount of one or more of the compoundsdisclosed herein. In certain embodiments, the pharmaceuticalcompositions are useful for treating inflammation, particularlycytokine-induced inflammation. The therapeutically effective amount of adisclosed compound will depend on the route of administration, thespecies of subject and the physical characteristics of the subject beingtreated. Specific factors that can be taken into account include diseaseseverity and stage, weight, diet and concurrent medications. Therelationship of these factors to determining a therapeutically effectiveamount of the disclosed compounds is understood by those of skill in theart.

Pharmaceutical compositions for administration to a subject can includeat least one further pharmaceutically acceptable additive such ascarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. The pharmaceutically acceptablecarriers useful for these formulations are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 19th Edition (1995), describes compositions and formulationssuitable for pharmaceutical delivery of the compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical compositions disclosed herein include those formed frompharmaceutically acceptable salts and/or solvates of the disclosedcompounds. Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Particular disclosed compounds possess at least one basic group that canform acid-base salts with acids. Examples of basic groups include, butare not limited to, amino and imino groups. Examples of inorganic acidsthat can form salts with such basic groups include, but are not limitedto, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuricacid or phosphoric acid. Basic groups also can form salts with organiccarboxylic acids, sulfonic acids, sulfo acids or phospho acids orN-substituted sulfamic acid, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconicacid, glucaric acid, glucuronic acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinicacid or isonicotinic acid, and, in addition, with amino acids, forexample with α-amino acids, and also with methanesulfonic acid,ethanesulfonic acid, 2-hydroxymethanesulfonic acid,ethane-1,2-disulfonic acid, benzenedisulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate or N-cyclohexylsulfamic acid(with formation of the cyclamates) or with other acidic organiccompounds, such as ascorbic acid. In particular, suitable salts includethose derived from alkali metals such as potassium and sodium, alkalineearth metals such as calcium and magnesium, among numerous other acidswell known in the pharmaceutical art.

Certain compounds include at least one acidic group that can form anacid-base salt with an inorganic or organic base. Examples of saltsformed from inorganic bases include salts of the presently disclosedcompounds with alkali metals such as potassium and sodium, alkalineearth metals, including calcium and magnesium and the like. Similarly,salts of acidic compounds with an organic base, such as an amine (asused herein terms that refer to amines should be understood to includetheir conjugate acids unless the context clearly indicates that the freeamine is intended) are contemplated, including salts formed with basicamino acids, aliphatic amines, heterocyclic amines, aromatic amines,pyridines, guanidines and amidines. Of the aliphatic amines, the acyclicaliphatic amines, and cyclic and acyclic di- and tri-alkyl amines areparticularly suitable for use in the disclosed compounds. In addition,quaternary ammonium counterions also can be used.

Particular examples of suitable amine bases (and their correspondingammonium ions) for use in the present compounds include, withoutlimitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane,diazabicycloundecene, N-methyl-N-ethylamine, diethylamine,triethylamine, diisopropylethylamine, mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additionalexamples of “pharmacologically acceptable salts,” see Berge et al., J.Pharm. Sci. 66:1 (1977).

Compounds disclosed herein can be crystallized and can be provided in asingle crystalline form or as a combination of different crystalpolymorphs. As such, the compounds can be provided in one or morephysical form, such as different crystal forms, crystalline, liquidcrystalline or non-crystalline (amorphous) forms. Such differentphysical forms of the compounds can be prepared using, for exampledifferent solvents or different mixtures of solvents forrecrystallization. Alternatively or additionally, different polymorphscan be prepared, for example, by performing recrystallizations atdifferent temperatures and/or by altering cooling rates duringrecrystallization. The presence of polymorphs can be determined by X-raycrystallography, or in some cases by another spectroscopic technique,such as solid phase NMR spectroscopy, IR spectroscopy, or bydifferential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by avariety of mucosal administration modes, including by oral, rectal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to other surfaces. Optionally, the compositions can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-arterial, intra-articular,intraperitoneal, intrathecal, intracerebroventricular, or parenteralroutes. In other alternative embodiments, the compound can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a subject.

To formulate the pharmaceutical compositions, the compound can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the compound. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubilityenhancing agents (for example, cyclodextrins and derivatives thereof),stabilizers (for example, serum albumin), and reducing agents (forexample, glutathione) can be included. Adjuvants, such as aluminumhydroxide (for example, Amphogel, Wyeth Laboratories, Madison, N.J.),Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), amongmany other suitable adjuvants well known in the art, can be included inthe compositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The compound can be combined with the base or vehicle according to avariety of methods, and release of the compound can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the compound is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al., J. Pharmacy Pharmacol.43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the compound can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe compound can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the compound can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the compoundand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, thecompound can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the compound and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

The administration of the compound of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, thecompound is provided in advance of any symptom. The prophylacticadministration of the compound serves to prevent or ameliorate anysubsequent disease process. When provided therapeutically, the compoundis provided at (or shortly after) the onset of a symptom of disease orinfection.

For prophylactic and therapeutic purposes, the compound can beadministered to the subject by the oral route or in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the compound can be provided as repeated doses within aprolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, avian, dog, sheep, porcine, feline, non-human primate, and otheraccepted animal model subjects known in the art. Alternatively,effective dosages can be determined using in vitro models. Using suchmodels, only ordinary calculations and adjustments are required todetermine an appropriate concentration and dose to administer atherapeutically effective amount of the compound (for example, amountsthat are effective to alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the compound may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the compound will vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the compound for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of a compound and/or other biologically active agent within themethods and formulations of the disclosure is about 0.01 mg/kg bodyweight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, intraosseous, or intranasal delivery versus intravenous orsubcutaneous or intramuscular delivery. Dosage can also be adjustedbased on the release rate of the administered formulation, for example,of an intrapulmonary spray versus powder, sustained release oral versusinjected particulate or transdermal delivery formulations, and so forth.

The compounds disclosed herein may also be co-administered with anadditional therapeutic agent. Such agents include, but are not limitedto, another anti-inflammatory agent, an antimicrobial agent, a matrixmetalloprotease inhibitor, a lipoxygenase inhibitor, a cytokineantagonist, an immunosuppressant, an anti-cancer agent, an anti-viralagent, a cytokine, a growth factor, an immunomodulator, a prostaglandinor an anti-vascular hyperproliferation compound.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of diseases and other conditions inmammalian subjects. Kits for diagnostic use are also provided. In oneembodiment, these kits include a container or formulation that containsone or more of the compounds described herein. In one example, thiscomponent is formulated in a pharmaceutical preparation for delivery toa subject. The compound is optionally contained in a bulk dispensingcontainer or unit or multi-unit dosage form. Optional dispensing meanscan be provided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

Results

FBXL2 targets TRAFs for polyubiquitination. It was presently observedthat ectopic expression of FBXL2 in murine lung epithelia (MLE)specifically reduces TRAF1-6 protein levels and phosphorylation levelsof the p105 subunit in the NF-κb pathway (FIG. 1A). FBXL2 was alsoconditionally expressed in MLE cells using a doxycycline-inducibleplasmid resulting in TRAF protein degradation in a time-dependent manner(FIG. 1B). In coimmunoprecipitation experiments where cells were lysedand subjected to FBXL2 immunoprecipitation (i.p.), all TRAF proteinswere detected in FBXL2 immunoprecipitates by immunoblotting (FIG. 1C).The results suggest that FBXL2 interacts with TRAFs in cells.Importantly, inclusion of purified SCFFBXL2 with the full complement ofE1 and E2 enzymes plus ubiquitin was sufficient to generatepolyubiquitinated TRAF species in vitro (FIG. 1D). Lastly, ectopicexpression of FBXL2 decreased TRAF protein half-life (FIG. 1E) but nottheir mRNA levels (data not shown).

FBXL2 is polyubiquitinated at the lysine 201 site.Since FBXL2 is animportant regulator of TRAFs, the mechanism involved in FBXL2 stabilityand degradation was investigated. First, several FBXL2 deletion mutantslacking specific lysine ubiquitin acceptor sites (FIG. 2A, top map) wereconstructed, and their vulnerability to polyubiquitination was tested byexposing cells to the 26S proteasome inhibitor MG132. Full length (FL)and four other FBXL2 deletion mutants all displayed significantaccumulation of high molecular weight ubiquitination products (FIG. 2A,bottom, right). Further deletional analysis suggested that a FBXL2-C150mutant is resistant to ubiquitination as no significant accumulation ofslower migrating species were detected (FIG. 2B, bottom right). Thereare two potential ubiquitination sites within 50 residues between FBXL2C150 and C200. Site-directed mutagenesis of these sites and expressionof a plasmid encoding these mutants resulted in significant resistanceof the FBXL2 K201R mutant to the 26S proteasome inhibitor MG132 (FIG.2C). The stability of this mutant was also tested in a half-life(t_(1/2)) study, which indicated significantly prolonged t_(1/2)compared to WT FBXL2 (2.5 h, FIG. 2D).

FBXL2 is phosphorylated and targeted by the SCF E3 ligase subunit FBXO3at residue T404.SCF-based E3 ligases target phosphoproteins. Databaseanalysis indicates many potential phosphorylation sites within the FBXL2(FIG. 3A, GPS2.1 software prediction). To confirm that FBXL2 isphosphoprotein, cells were lysed and subjected to FBXL2 i.p., and usingphospho-threonine antibodies we were able to detect a band whichmigrates at the predicted size of FBXL2 (FIG. 3B). In order to identifythe potential kinase that targets FBXL2 for phosphorylation, weperformed co-immunoprecipitation (co-i.p.) experiments. MLE cells werelysed and subjected to FBXL2 i.p.; interestingly, out of seven kinasestested, GSK3J was the only protein detected in the FBXL2immunoprecipitates (FIG. 3C). Because FBXL2 is a phosphoprotein thatmight be targeted for SCF-based ubiquitination, we started an unbiasedscreen randomly testing F-box proteins that might mediate FBXL2degradation. Upon overexpression of these proteins, only FBXO3 was ableto decrease the levels of immunoreactive FBXL2 (data not shown). FBXO3belongs to a large group of F-box proteins lacking a distinct C-terminalmotif, thus deemed F-box domain only proteins (FBXOs). Only one studyshowed that FBXO3 increases ubiquitination of p300, and its authenticityas an SCF subunit and its substrates remain largely unknown. To confirmthe specificity of FBXO3 targeting FBXL2, co-i.p. experiments wereperformed where FBXO3 was detected in the FBXL2 immunoprecipitates (FIG.3D).

Further, the SCFFBXO3 complex was able to induce polyubiquitination ofFBXL2 (FIG. 3E). Using the FBXL2 deletion mutants described in FIG. 2A,preliminary mapping studies transfecting cells with histagged FBXL2constructs followed by his-pull down were performed. Our resultsindicate that FBXO3 docks at the C-terminus (residues 350-423) of FBXL2(FIG. 3F). To confirm that this region is important for FBXL2 stability,wild-type (WT) FBXL2 and several FBXL2 C-terminal deletion mutants weretested for stability (FIG. 3G). Interestingly, a FBXL2 C390 deletionmutant exhibited significantly prolonged t_(1/2) compared WT FBXL2,suggesting that residues 390-423 are important for its stability. Withinthis region, there is a consensus GSK3β phosphorylation site (FIG. 3H,GPS2.1 software prediction). To confirm that T404 is the authentic FBXL2phosphorylation site, cells transfected with either a WT FBXL2 or FBXL2T404A mutant were lysed and subjected to V5-FBXL2 i.p., andimmunoblotted using phospho-threonine antibodies where a significantdecrease in FBXL2 T404A protein phosphorylation levels was detected(FIG. 3I). Interestingly, this site also serves as a targeting motif forFBXO3 interaction, as FBXL2 T404A exhibits significant resistance toSCFFBXO3 using in vitro ubiquitination assays (FIG. 3J). In summary,FBXO3 targets a T404 phosphorylation site within FBXL2, which in turnrecruits the SCF complex to polyubiquitinate FBXL2 at a K201 site (FIG.3K).

FBXO3 contains a natural occurring mutation at V220. Interestingly, theSNP database analysis indicates a natural occurring mutation withinFBXO3 (Val220Ile) with a very high mutation frequency of ˜10%, thoughonly in Caucasians (FIG. 4A). To confirm that V220I is a relevant FBXO3mutation in human cells, PBMC samples from twenty healthy Caucasianvolunteers (commercially available through Sanguine Life Science) wereanalyzed. Genomic DNA was first extracted from PBMC cells followed bySNP genotyping through TaqMan® SNP probe using real-time PCR. ThreeCaucasian PBMC samples harboring FBXO3V220I mutations were identified(FIG. 4B). These PBMC cells containing this FBXO3 mutation were testedusing in vitro assays for cytokine release. Wt or mutant PMBC cells werefirst cultured in RPMI medium supplemented with 10% FBS, cells were thentreated with 2 ug/ml LPS for 24 h, and cytokines released in the mediumwere assayed using a human cytokine array. Interestingly, in the LPSinduced model, the induction of several major pro-inflammatory cytokineswere significantly suppressed in PBMC cells harboring the FBXO3V220Imutation compared to WT PBMC cells (FIG. 4C); thus, the FBXO3V220Imutation might confer a reduced pro-inflammatory phenotype in subjectswith infection or other autoimmune diseases.

The nature of the FBXO3 V220I mutation was subsequently tested. Comparedto WT FBXO3, the SCF-FBXO3V220I complex displayed markedly reducedability to polyubiquitinate FBXL2 with most of the substrate intact(FIG. 4D, below, lighter exposure). FBXO3 function in U937 monocytes wasthen studied, which adopt the morphology and many characteristics ofmature macrophages. Preliminary data show that FBXL2 ubiquitinates andmediates degradation of TRAF proteins thereby potentially reducingcytokine expression. Thus, by eliminating FBXL2 in cells, it ishypothesized that FBXO3 should be able to up-regulate TRAF proteinlevels and stimulate cytokine expression. Indeed, consistent with thishypothesis, FBXO3 overexpression was able to decrease FBXL2 proteinlevels, yet significantly increase all six TRAF protein member levels(FIG. 4E). However, overexpression of FBXO3V220I only resulted in basalor no increase in several TRAF proteins. U937 cell cytokine release uponLPS challenge was further monitored. Cells were first transfected withLacZ, FBXO3, or FBXO3V220I for 24 h before exposure to LPS at 100 ng/mlfor an additional 24 h. Thirty six cytokines levels were measured usinga human cytokine array. Interestingly, it was observed that FBXO3significantly up-regulates most of the cytokines released in combinationwith LPS challenge (FIG. 4F, red); however, FBXO3V220I expression didnot dramatically alter cytokine release compared to the LacZ control(FIG. 4F). These novel results are the first linking two F-box proteinsto the innate immune response and suggest that FBXO3V220I is a loss-offunction mutation of FBXO3. The results raise the possibility thatindividuals that harbor this naturally occurring hypomorphic mutationmight exhibit a blunted response to infection or other auto-immunediseases.

FBXO3V220I is a loss-of-function mutation of FBXO3 in vivo. To extendthe above observations in vivo, mice were infected with an emptylentivirus, or lentivirus encoding either FBXO3 or FBXO3V220I for 120 h(10⁷ CFU/mouse, i.t.). Mice were then challenged with P. aeruginosa(strain PA103, 10⁴ CFU/mouse, i.t.) for an additional 24 h. Mice werethen monitored with FlexiVent to measure lung mechanics and euthanizedto collect lavage fluid. Wt FBXO3 expression, but not FBXO3V220I,significantly augmented PA103 induced lung injury. Specifically, FBXO3overexpression significantly increased lung resistance and elastance,and decreased compliance (FIG. 5A-D). FBXO3 overexpression significantlyincreased lavage protein concentration, lavage cell counts and cellinfiltrates (FIG. 5E-G). FBXO3 also decreased survival of PA103 infectedmice (10⁵ CFU/mouse, FIG. 5H). FBXO3 overexpression also significantlyincreased lavage cytokine levels in PA103 infected mice compared toempty vector with or without PA103 (FIG. 5I). These effects were notobserved using the FBXO3V220I mutant. These in vivo studies suggestagain that FBXO3V220I is a loss-of-function mutant of FBXO3.

FBXO3 knockdown ameliorated pseudomonas induced lung injury in vivo. Toconfirm the role of FBXO3 in pneumonia, in vivo knockdown studies werepursued where mice were infected with lentivirus encoding empty shRNA orFBXO3 shRNA for 120 h (10⁷ PFU/mouse, i.t). Mice were then challengedwith PA103 (10⁴ CFU/mouse, i.t.) for an additional 24 h. Interestingly,FBXO3 knockdown significantly ameliorated adverse effects of PA103 onlung mechanics. Specifically, FBXO3 knockdown increased compliance,decreased lung resistance and elastance (FIG. 6A-D). FBXO3 knockdownalso decreased lavage protein concentration, lavage cell counts and cellinfiltrates (FIG. 6E-G). Further, FBXO3 knockdown significantlydecreased lavage cytokine levels in PA103 infected mice (FIG. 6H) andincreased their survival (10⁵ CFU/mouse, FIG. 6I). These in vivo studiessuggest that FBXO3 plays an important role in regulating the cytokinestorm and may serve as a potential pharmaceutical target. Thus, toinvestigate the potential application of FBXO3 inhibition in pneumonia,the FBXO3 structure was analyzed and small molecule inhibitors werescreened.

FBXO3 ApaG domain structural analysis and inhibitor screening. FBXO3harbors a very unique domain termed ApaG within its carboxyl-terminus.The ApaG domain was first identified in bacteria, containing ˜125 aminoacids, which comprises a core. However, the function of the ApaG proteinin bacteria is unknown. In Salmonella typhimurium, the ApaG domainprotein, CorD, is involved in Co2+ resistance and Mg2+ efflux.Structural analysis from different ApaG proteins shows a fold of severalbeta-sheets. Since F-box proteins often utilize their carboxyl-terminaldomain to target their substrates, it was hypothesized that the FBXO3ApaG domain is involved in FBXO3 substrate recognition. To test this, aseries of FBXO3 deletion mutants was designed where the ApaG domain wasdeleted (FIG. 7A). In vitro transcription and translation (TnT) wereused to synthesize these mutants and which were then tested in the invitro ubiquitination assay using FBXL2 as the substrate. Interestingly,FBXO3-C278, which lacks the ApaG domain, lost the ability to inducepolyubiquitination of FBXL2 (FIG. 7B); FBXO3-N70, which lacks theNH₂-terminal F-box domain required to interact with the SCF complex,served as a negative control. These experiments suggest that theFBXO3-ApaG domain is required for FBXL2 targeting. Next it washypothesized that inhibition of the ApaG domain disrupts FBXO3 targetingto its substrate, FBXL2. A structural homology analysis was performedidentifying that the FBXO3-ApaG domain is highly conserved (FIG. 7C).Using molecular docking analysis and scored-ranking operations on thepredicted FBXO3-ApaG 3-D structure model, potential ligands wereassessed that might fit the ApaG domain cavities (FIG. 7D). The dockingexperiments were carried out by using LigandFit and CDock from Discoverystudio 2.5. A library containing 6507 approved or experimental drugswere first used to screen potential ligands for FBXO3-ApaG. In thismodel, Glu⁶⁴ within the ApaG domain (123AA) is potentially important forinteracting inhibitors. Based on the docking and best-fit analysis ofsuitable ligands, benzathine was selected as a backbone to develop aseries of new biomolecules to test their abilities to inhibit cytokinesecretion by interacting within the ApaG binding pocket (FIG. 7E-F).

FBXO3 inhibitors preparation and docking analysis. The target benzathineanalogs were prepared from benzaldehyde derivatives and diaminederivatives such as ethylenediamine (FIG. 8A). In general, the relevantbenzaldehyde derivatives (0.02 mol) were added to a solution ofethylenediamine (0.01 mol, ˜700 ul) in anhydrous ethanol (20 ml). Theresulting solution was refluxed and stirred for 60 min until theprecipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitates of benzathine analogswere collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

As shown in table 1, forty new compounds were constructed and tested fortheir IC50, LD50 and therapeutic index (TI). Briefly, compounds wereadded to human PBMC cells at different concentrations that were exposedto LPS and cytokine secretion was monitored by ELISA to determine theIC50. Compounds were also added to U937 monocytes at differentconcentrations, and cells were stained with trypan blue to determine theLD50. Several compounds (BC-1207, BC-1215, BC-1241, BC-1250 and BC-1261)scored high in docking studies with the FBXO3-ApaG domain and exhibitedhigh IC50 and low LD50 in vitro. Importantly, several new smallmolecules, termed BC-1215 and BC-1261, exhibited optimal interactionswith FBXO-ApaG based on structural and docking analysis as shown in FIG.8B-D. These specific agents exhibited remarkable therapeutic indicesthat warranted further biological testing. Several functional studieswere undertaken to assess anti-inflammatory effects focusing on BC-1215.

BC-215 profoundly inhibits a broad spectrum of cytokines. PBMC cellswere treated with 2 ug/ml LPS for 16 hrs along with BC-1215 at 10 ug/ml.Cytokine release was monitored by a human cytokine array (R&D systems).The results from FIG. 9 indicate remarkable ability of BC-1215 tosignificantly suppress the majority of the TH1 panel cytokines includingG-CSF, GM-CSF, GROα, I-309, IL1-α, IL1-β, IL1rα, IL-6, IL-12, IL-23,MIP-1α, MIP-1β and TNFα. These cytokines are tightly linked to thepathogenesis of many pro-inflammatory diseases, some of which have ledto the use of blocking cytokine antibodies to reduce disease severity.For example: GM-CSF drives inflanmmation in rheumatoid arthritis (RA),and currently, GM-CSF blocking antibodies (MOR103) have been tested inPhase 1b/2a trial in patients suffering from RA.

Canakinumab, a human IL1-β blocking antibody has been approved fortreatment of cryopyrin-associated periodic syndromes and is being testedin Phase 1 trials for chronic obstructive pulmonary disease. IL-6 hasbeen linked to many auto-immune diseases and cancer, and recently IL-6blocking antibody was tested in Phase 2 trials in patients sufferingfrom non-small cell lung cancer. IL-12 and IL-23 are linked withautoimmunity; Ustekinumab (commercial name Stelara) is a humanmonoclonal antibody against IL-12 and IL-23, which has been approved totreat moderate to severe plaque psoriasis. TNFα, a critical TH1cytokine, also promotes the inflammatory response, and is etiologicallylinked to many autoimmune disorders such as RA, inflammatory boweldisease, psoriasis, and refractory asthma. Several TNFα blockingantibodies such as infliximab (Remicade), adalimumab (Humira) orcertolizumab (Cimzia) have been approved to treat these autoimmunedisorders. However, many of the above approaches have a limited spectrumof bioactivity as they target a single cytokine and are directed againsta host protein. The data disclosed herein are significant in that theysuggest that this new family of F box protein E3 ligase antagonists(e.g. BC-1215) described herein may be more efficacious in inflammatorydisorders as they are panreactive to several pro-inflammatory moleculesand they target a unique bacterial-like molecular signature in hostcells. These unique properties of F box protein E3 ligase antagonistswill confer greater anti-inflammatory activities and yet have limitedoff-target effects.

BC-1215 inhibits FBXO3 and decreases TRAF protein levels. To establish amechanistic link between infection and cytokine release, PBMC cells weretreated with LPS, and downstream signaling proteins were assayed byimmunoblotting. It was found that LPS increases FBXO3 protein levels,decreases FBXL2 protein levels, and increases TRAF protein levels (FIG.10A). Thus, pro-inflammatory signaling by endotoxin actions might bemediated though FBXO3 protein. BC-1215 was first tested in in vitroubiquitination assays using FBXL2 as substrate. BC-1215 was able toinhibit FBXO3 catalyzed FBXL2 polyubiquitination (FIG. 10B). MLE cellswere also treated with BC-1215 at different concentrations for 16 h.Cells were collected and assayed for protein immunoblotting. As shown inFIG. 10C, BC-1215 increased FBXL2 protein levels in a dose dependentmanner, in turn decreasing TRAF protein levels. Other known FBXL2substrates including cyclin D2, cyclin D3, and CCTalpha served aspositive controls. We also observed that BC-1215 did not significantlyalter cell cycle progression of Hela cells in the therapeutic doses(FIG. 10D). BC-1215 did not alter COX-2 activity compared to thepositive control, DuP-697 (FIG. 10E). These latter results stronglysuggest that BC-1215 and related agents mechanistically represent a newgenus of anti-inflammatories that exerts activities independent ofmechanisms used by nonsteroidal anti-inflammatory drugs (NSAIDs) whichact as COX-2 inhibitors. Based on the novel mechanism of action ofBC-1215, the effectiveness of this agent was tested in several differentinflammation models in mice.

BC-1215 potently inhibits cytokine release in a LPS induced septic shockmodel. Compound BC-1215 was first solubilized in water using acetic acidin a 1:2 molar ratio; the stock solution of BC-1215 was 5 mg/ml. 500 ug,100 ug, 20 ug, 4 ug and 0.8 ug of BC-1215 was administered to micethough an intraperitoneal (IP) injection. 10 min later, mice were given100 ug of LPS (E. coli) through an IP injection. 90 min later, mice wereeuthanized; blood was collected and tested for IL1-β, IL-6 and TNFα,cytokine assays. The results from FIG. 11 indicate that IC50_(IL-1β)32 1mg/kg, IC50_(IL-6)=2.5 mg/kg, IC50_(TNFα)=1.2 mg/kg. These IC50s areconsidered very low considering that the predicted mouse oral LD50 dosefor BC-1215 is 1.135 g/kg; thus BC-1215 exerts bioactivity well below apredicted toxic dose in vivo.

BC-1215 inhibits cytokine release in a cecal ligation and puncture (CLP)sepsis model. Compound BC-1215 was first solubilized as above. 100 ug ofBC-1215 was administered to mice though an IP injection. 30 min later,CLP was performed. 6 h later, mice were euthanized; blood was collectedand tested for IL1-β, IL-6 and TNFα cytokines. As shown in FIG. 12, CLPtreated mice had significantly increased cytokine release compared tosham treated mice. However, BC-1215 was able to significantly attenuateCLP-induced secretion of all three circulating pro-inflammatorycytokines in mice.

BC-1215 reduces lung injury in pseudomonas induced pneumonia. To testthe F box inhibitor BC-1215 in pneumonia, 100 ug of BC-1215 wasadministered to mice though an IP injection, mice were then challengedwith Pseudomonas aeruginosa strain PA103 (10⁴ CFU/mouse, i.t.) for anadditional 18 h. Interestingly, BC-1215 significantly amelioratedadverse effects of PA103 on lung mechanics. Specifically, BC-1215increased compliance, decreased lung resistance, and reduced elastance(FIG. 13A-D). BC-1215 also decreased lavage protein concentration,lavage cell counts and cell infiltrates (FIG. 13E, F, G). Further,BC-1215 also significantly decreased lavage pro-inflammatory cytokinelevels in PA103 infected mice (FIG. 13H).

BC-1215 ameliorates H1N1 influenza induced lung injury in vivo. Tofurther test BC-1215 in pneumonia, mice were challenged with H1N1 (10⁵PFU/mouse, i.t.) and observed for 9 d. For BC-1215 treatment, a stocksolution (5 mg/ml) was added to drinking water (containing 2% sucrose)to the final concentration of 30 ug/ml. Lung mechanics was measured atday 5. Specifically, BC-1215 increased compliance, decreased lungresistance and reduced elastance (FIG. 14A-C) in mice infected withH1N1. Further, BC-1215 significantly increased their survival with H1N1pneumonia (FIG. 14D). BC-1215 also remarkably decreased lavage proteinconcentration, lavage cell counts (FIG. 14E, F), lung edema and cellinfiltrates (FIG. 14G, H).

BC-1215 reduces TPA induced ear edema. Topical application of BC-1215 asan anti-inflammatory agent was tested in a model of12-O-tetradecanoylphorbol-13-acetate (TPA) induced ear edema (Bralleyet. al., J Inflamm (LOnd), 2008. 5:p. 1). Briefly, 20 μl of an ethanolsolution of BC-1215 was applied to ears of mice at 8, 40, and 200 ug/earfor 30 min after TPA administration (2 μg/ear). Comparisons includedequal volumes of ethanol (vehicle control). 18 h after TPAadministration, mice were euthanized; the thickness of the ear wasmeasured using a micrometer. Ear punch biopsies were also takenimmediately, weighed, and graphed. As shown in FIG. 15A, ear edema wasobserved in the TPA-treated animals at 18 h after its treatment.However, BC-1215 was able to significantly resolve edema. As shown inFIG. 15B—C, BC-1215 significantly reduced ear thickness and ear weightin a dose dependent manner compared to the vehicle control. Thesestudies demonstrate for the first time that the FBXO3 inhibitor BC-1215,by inhibiting development of edema, may have topical applicability andthus may have a role in dermatologic inflammatory disorders.

BC-1215 ameliorates carrageenan induced paw edema. BC-1215 also wastested in a mouse paw edema model to confirm its anti-inflammatoryactivity. Mice received subplantar administration of 25 ul of saline or25 ul of carrageenan (1% in saline) (Posadas et al., Br J Pharmacol,2004. 142(2):p. 331-8), followed by an IP injection of 200 ug of BC-1215daily. 48 h later, mice were euthanized; the thickness and volume of thepaw was measured. As shown in FIG. 16A, paw edema was observed incarrageenan-treated animals at 48 h. However, BC-1215 was able tosignificantly suppress this affect. As shown in FIG. 16B—C, BC-1215significantly reduced paw thickness and edema compared to vehiclecontrol. Thus, the FBXO3 inhibitor BC-1215 suppresses inflammation in anonpulmonary model of edema involving the extremities.

BC-1215 ameliorates DSS induced colitis. BC-1215 was also tested in amouse colitis model to confirm its anti-inflammatory activity. Briefly,C57BL6 mice were fed with water containing 3.5% dextran sulfate sodium(DSS) for up to five days. Mice were treated with either vehicle or 200ug of BC-1215 daily (via IP injection). Mice were then euthanized; thelength of colons was measured. As shown in FIG. 17A, a significantdecrease in colon length was observed with mice treated with DSS,consistent with colonic inflammation. However, mice treated with BC-1215shown no significant decrease in colon length compared to control.Colonic tissue cytokine levels were analyzed. As shown in FIG. 17B-C,mice treated with BC-1215 showed a remarkable reduction in IL13 and TNFαlevels in colon tissues compared to vehicle treated mice. Further,BC-1215 significantly reduced colonic tissue injury in DSS treated mice(FIG. 17D). Thus, the FBXO3 inhibitor BC-1215 suppresses inflammation inchemical induced colitis model in mice.

In summary, disclosed herein is the first evidence in any system thatinflammation is mediated in part, by a novel pathway whereby apreviously unrecognized E3 ligase component, FBXO3, triggersubiquitination and degradation of another E3 ligase subunit, FBXL2,thereby increasing levels of TRAF proteins. In essence, FBXL2 appears tobe a feedback inhibitor of inflammation. As TRAFs are critical molecularinputs to NF-κB-driven cytokine gene expression, abrogation of FBXO3 isable to prevent induction of TRAF proteins and suppress cytokineproduction (FIG. 18). Hence, based on the unique molecular structure ofFBXO3 as the centerpiece of this discovery, a new phylum of F boxubiquitin-E3 ligase based ApaG small molecule inhibitors was generatedthat profoundly exert anti-inflammatory activity in human cells and incomplementary small animal models of tissue inflammation and injury.

BC-1215 inhibits S. aureus proliferation. BC-1215 was tested inantibiotic sensitivity tests using Mueller-Hinton agar as shown in FIG.19. Briefly, 6 mm filter papers containing different amounts of BC-1215or gentamicin antibiotic (positive control) were added on theMueller-Hinton agar pre-exposed to Staphylococcus aureus. The plateswere incubated at 37 degrees for 24 h. Zone sizes were measured andmarked by a red circle indicating positive results. The data heresuggests that BC-1215 may inhibit bacterial growth through the bacterialApaG protein.

FBXO3 Inhibitors Synthesis

General procedure for synthesis of BC-1202. 4-(Benzyl-Oxy)Benzaldehyde(0.01 mol, 2.12 g) were added to a solution of ethylenediamine (0.005mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 20 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 30 ml absolute methanol. A10% solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1202 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1203.4-(Dimethylamino)Benzaldehyde (0.01 mol, 1.49 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1203 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1204. 4-Methoxy-benzaldehyde (0.02mol, 2.72 g) were added to a solution of ethylenediamine (0.01 mol, ˜700ul) in anhydrous ethanol (40 ml). The resulting solution was heated andstirred for 40 min until the precipitation of the relevant Schiff base.The Schiff bases were filtered off, and washed with cold ethanol. TheSchiff base was then added to 30 ml absolute methanol. A 10% solution ofsodium borohydride (0.02 mol) was dissolved in absolute methanol andadded to the Schiff base. When the dropwise addition of sodiumborohydride was complete, the reaction solution was refluxed for anadditional 15 min. Solvent was then removed through rotary evaporationand 40 ml cold water was added to liberate the secondary amine. Theproduct BC-1204 was then extracted with EtOAC and the organic layerwashed with water, dried over Na2SO4 and concentrated under vacuum.

General procedure for synthesis of BC-1205.4-(4-Morpholinyl)benzaldehyde (0.01 mol, 1.91 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1205 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1206.4-(1-Pyrrolidino)-benzaldehyde (0.01 moL 1.75 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1206 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1207.4-(1H-Imidazol-1-yl)benzaldehyde (0.01 mol, 1.72 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1207 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1208. 4-Acetylbenzaldehyde (0.01mol, 1.48 g) were added to a solution of ethylenediamine (0.005 mol,˜350 ul) in anhydrous ethanol (20 ml). The resulting solution wasrefluxed and stirred for 60 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 30 ml absolute methanol. A10% solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1208 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1209. 2-Hydroxybenzaldehyde (0.01mol, 1.22 g) were added to a solution of ethylenediamine (0.005 mol,˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heatedand stirred for 10 min until the precipitation of the relevant Schiffbase. The Schiff bases were filtered off, and washed with cold ethanol.The Schiff base was then added to 30 ml absolute methanol. A 10%solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1209 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1210. 4-Hydroxybenzaldehyde (0.01mol, 1.22 g) were added to a solution of ethylenediamine (0.005 mol,˜350 ul) in anhydrous ethanol (20 ml). The resulting solution was heatedand stirred for 10 min until the precipitation of the relevant Schiffbase. The Schiff bases were filtered off, and washed with cold ethanol.The Schiff base was then added to 30 ml absolute methanol. A 10%solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1210 were collected, washed with waterand dried, followed by recrystallization from ethanol.

General procedure for synthesis of BC-1211.4-Trifluoromethoxy)benzaldehyde (0.01 mol. 1.9 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 60 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The product BC-1211 was then extractedwith EtOAC and the organic layer washed with water, dried over Na2SO4and concentrated under vacuum.

General procedure for synthesis of BC-1212.4-(Dimethylamino)benzaldehyde (0.01 mol, 1.49 g) were added to asolution of 1,2-Phenylenediamine (0.005 mol, 0.54 g) in anhydrousethanol (20 ml). The resulting solution was heated and stirred for 30min. The reaction was cooled down until the precipitation of therelevant Schiff base. The Schiff bases were filtered off, and washedwith cold ethanol. The Schiff base was then added to 30 ml absolutemethanol. A 10% solution of sodium borohydride (0.02 mol) was dissolvedin absolute methanol and added to the Schiff base. When the dropwiseaddition of sodium borohydride was complete, the reaction solution wasrefluxed for an additional 15 min. Solvent was then removed throughrotary evaporation and 40 ml cold water was added to liberate thesecondary amine. The precipitation of BC-1212 were collected, washedwith water and dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1213.4-(Dimethylamino)benzaldehyde (0.01 mol, 1.49 g) were added to asolution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) inanhydrous ethanol (20 ml). The resulting solution was heated and stirredfor 20 min until the precipitation of the relevant Schiff base. TheSchiff bases were filtered off, and washed with cold ethanol. The Schiffbase was then added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1213were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1214.4-(1-Piperidinyl)benzaidehlyde (0.01 mol, 1.89 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was refluxed and stirred for 30 minuntil the precipitation of the relevant Schiff base. The Schiff baseswere filtered off, and washed with cold ethanol. The Schiff base wasthen added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1214were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1215. 4-(2-Pyridinyl)benzaldehyde(0.01 mol, 1.83 g) were added to a solution of ethylenediamine (0.005mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 30 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 30 ml absolute methanol. A10% solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1215 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1216. 3,4,5-Trimethoxybenzaldehyde(0.01 mol, 1.96 g) were added to a solution of ethylenediamine (0.005mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 30 min. The reaction was cooled down until theprecipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1216 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1217.4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to asolution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) inanhydrous ethanol (20 ml). The resulting solution was heated and stirredfor 20 min until the precipitation of the relevant Schiff base. TheSchiff bases were filtered off, and washed with cold ethanol. The Schiffbase was then added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1217were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1218.4-(1-Piperidinyl)benzaldehyde (0.01 mol, 1.89 g) were added to asolution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) inanhydrous ethanol (20 ml). The resulting solution was heated and stirredfor 20 min until the precipitation of the relevant Schiff base. TheSchiff bases were filtered off, and washed with cold ethanol. The Schiffbase was then added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1218were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1220.4-(4-Morpholinyl)benzaldehyde (0.01 mol, 1.91 g) were added to asolution of (+/−)-trans-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) inanhydrous ethanol (20 ml). The resulting solution was heated and stirredfor 20 min until the precipitation of the relevant Schiff base. TheSchiff bases were filtered off, and washed with cold ethanol. The Schiffbase was then added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1220were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1232.4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to asolution of 1,2-Phenylenediamine (0.005 mol, 0.54 g) in anhydrousethanol (20 ml). The resulting solution was refluxed and stirred for 30min. The reaction was cooled down until the precipitation of therelevant Schiff base. The Schiff bases were filtered off, and washedwith cold ethanol. The Schiff base was then added to 30 ml absolutemethanol. A 10% solution of sodium borohydride (0.02 mol) was dissolvedin absolute methanol and added to the Schiff base. When the dropwiseaddition of sodium borohydride was complete, the reaction solution wasrefluxed for an additional 15 min. Solvent was then removed throughrotary evaporation and 40 ml cold water was added to liberate thesecondary amine. The precipitation of BC-1232 were collected, washedwith water and dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1233.4-(1-Pyrrolidino)-benzaldehyde (0.01 moL. 1.75 g) were added to asolution of (1S,2S)-(+)-1,2-Diaminocyclohexane (0.005 mol, 0.57 g) inanhydrous ethanol (20 ml). The resulting solution was heated and stirredfor 20 min until the precipitation of the relevant Schiff base. TheSchiff bases were filtered off, and washed with cold ethanol. The Schiffbase was then added to 30 ml absolute methanol. A 10% solution of sodiumborohydride (0.02 mol) was dissolved in absolute methanol and added tothe Schiff base. When the dropwise addition of sodium borohydride wascomplete, the reaction solution was refluxed for an additional 15 min.Solvent was then removed through rotary evaporation and 40 ml cold waterwas added to liberate the secondary amine. The precipitation of BC-1233were collected, washed with water and dried, followed byrecrystallization from ethyl acetate.

General procedure for synthesis of BC-1234.4-(1-Pyrrolidino)-benzaldehyde (0.01 mol, 1.75 g) were added to asolution of 1,4-Diaminobutane (0.005 mol, 0.44 g) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1234 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1239.4-(1-Pyrrolidino)-benzaldehyde (0.01 moL. 1.75 g) were added to asolution of 1,3-Diaminopropane (0.005 mol, 0.37 g) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1239 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1241. 4-(2-Pyridinyl)benzaldehyde(0.005 mol, 0.92 g), 4-fluorobenzaldehyde (0.005 mol, 0.62 g) were addedto a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrousethanol (20 ml). The resulting solution was refluxed and stirred for 60min. The reaction was cooled down until the precipitation of therelevant Schiff base. The Schiff bases were filtered off, and washedwith cold ethanol. The Schiff base was then added to 30 ml absolutemethanol. A 10% solution of sodium borohydride (0.02 mol) was dissolvedin absolute methanol and added to the Schiff base. When the dropwiseaddition of sodium borohydride was complete, the reaction solution wasrefluxed for an additional 15 min. Solvent was then removed throughrotary evaporation and 40 ml cold water was added to liberate thesecondary amine. The precipitation of BC-1241 were collected, washedwith water and dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1248. 4-(2-Pyridinyl)benzaldehyde(0.005 mol, 0.92 g), 2-Pyridinecarboxaldehyde (0.005 mol, 0.53 g) wereadded to a solution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrousethanol (20 ml). The resulting solution was refluxed and stirred for 60min. The reaction was cooled down until the precipitation of therelevant Schiff base. The Schiff bases were filtered off, and washedwith cold ethanol. The Schiff base was then added to 30 ml absolutemethanol. A 10% solution of sodium borohydride (0.02 mol) was dissolvedin absolute methanol and added to the Schiff base. When the dropwiseaddition of sodium borohydride was complete, the reaction solution wasrefluxed for an additional 15 min. Solvent was then removed throughrotary evaporation and 40 ml cold water was added to liberate thesecondary amine. The precipitation of BC-1248 were collected, washedwith water and dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1250.4-(1H-Pyrazol-1-yl)benzaldehyde (0.004 mol, 0.7 g) were added to asolution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol(10 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 15 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 20 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1250 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1251.5-Chloro-2-Hydroxybenzaldehyde (0.01 mol, 1.56 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1251 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1252.2-Hydroxy-4-Methoxybenzaldehyde (0.01 mol, 1.52 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1252 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1253. 2,4-Dihydroxybenzaldehyde(0.01 inol, 1.38 g) were added to a solution of ethylenediamine (0.005mol, ˜350 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 20 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 30 ml absolute methanol. A10% solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1253 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1254. 4-(2-Pyridinyl)benzaldehyde(0.01 mol, 1.83 g) were added to a solution of 1,4-Diaminobutane (0.005mol, 0.44 g) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 20 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 30 ml absolute methanol. A10% solution of sodium borohydride (0.02 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 40 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1254 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1255. 4-(2-Pyridinyl)benzaldeyde(0.01 mol, 1.83 g) were added to a solution of 1,3-Diamino-2-Propanol(0.005 mol, 0.45 g) in anhydrous ethanol (20 ml). The resulting solutionwas heated and stirred for 20 min until the precipitation of therelevant Schiff base. The Schiff bases were filtered off, and washedwith cold ethanol. The Schiff base was then added to 30 ml absolutemethanol. A 10% solution of sodium borohydride (0.02 mol) was dissolvedin absolute methanol and added to the Schiff base. When the dropwiseaddition of sodium borohydride was complete, the reaction solution wasrefluxed for an additional 15 min. Solvent was then removed throughrotary evaporation and 40 ml cold water was added to liberate thesecondary amine. The precipitation of BC-1255 were collected, washedwith water and dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1256.2-(2-hydroxyethoxy)benzaldehyde (0.01 mol, 1.66 g) were added to asolution of ethylenediamine (0.005 mol, ˜350 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 40 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 30 ml absolute methanol. A 10% solution of sodium borohydride(0.02 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 40 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1256 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1257.4-Trifluoromethoxy)Salicaldehyde (0.004 moL 0.82 g) were added to asolution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 40 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 15 ml absolute methanol. A 10% solution of sodium borohydride(0.01 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 20 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1257 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1258.4-(1,3-Thiazol-2-yl)benzaldehyde (0.004 mol, 0.76 g) were added to asolution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 20 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 15 ml absolute methanol. A 10% solution of sodium borohydride(0.01 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 20 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1258 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1259. 4-(2-Thienyl)Benzaldehyde(0.004 mol, 0.76 g) were added to a solution of ethylenediamine (0.002mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 40 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 15 ml absolute methanol. A10% solution of sodium borohydride (0.01 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 20 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1259 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1260. 4-(2-furyl)benzaldehyde(0.004 mol. 0.69 g) were added to a solution of ethylenediamine (0.002mol, ˜140 ul) in anhydrous ethanol (20 ml). The resulting solution washeated and stirred for 40 min until the precipitation of the relevantSchiff base. The Schiff bases were filtered off, and washed with coldethanol. The Schiff base was then added to 15 ml absolute methanol. A10% solution of sodium borohydride (0.01 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 20 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1260 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

General procedure for synthesis of BC-1261.4-(pyrimidin-2-yl)benzaldehyde (0.004 mol, 0.74 g) were added to asolution of ethylenediamine (0.002 mol, ˜140 ul) in anhydrous ethanol(20 ml). The resulting solution was heated and stirred for 30 min untilthe precipitation of the relevant Schiff base. The Schiff bases werefiltered off, and washed with cold ethanol. The Schiff base was thenadded to 15 ml absolute methanol. A 10% solution of sodium borohydride(0.01 mol) was dissolved in absolute methanol and added to the Schiffbase. When the dropwise addition of sodium borohydride was complete, thereaction solution was refluxed for an additional 15 min. Solvent wasthen removed through rotary evaporation and 20 ml cold water was addedto liberate the secondary amine. The precipitation of BC-1261 werecollected, washed with water and dried, followed by recrystallizationfrom ethyl acetate.

General procedure for synthesis of BC-1262. 4-Phenylbenzaldehyde (0.004mol, 0.73 g) were added to a solution of ethylenediamine (0.002 mol,˜140 ul) in anhydrous ethanol (20 ml). The resulting solution was heatedand stirred for 20 min until the precipitation of the relevant Schiffbase. The Schiff bases were filtered off, and washed with cold ethanol.The Schiff base was then added to 15 ml absolute methanol. A 10%solution of sodium borohydride (0.01 mol) was dissolved in absolutemethanol and added to the Schiff base. When the dropwise addition ofsodium borohydride was complete, the reaction solution was refluxed foran additional 15 min. Solvent was then removed through rotaryevaporation and 20 ml cold water was added to liberate the secondaryamine. The precipitation of BC-1262 were collected, washed with waterand dried, followed by recrystallization from ethyl acetate.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention.

What is claimed is:
 1. A compound, or a pharmaceutically acceptable saltthereof, having a structure of formula VI:

wherein Ar¹ and Ar² are each independentaly optionally-substituted arlyor optionally-substituted N heterocyclic.
 2. The compound of claim 1,wherein Ar¹, Ar², or both Ar¹ and Ar², are selected from pyridyl,pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, morpholinyl,thiomorpholinyl, piperazinyl, or triazinyl.
 3. The compound of claim 1,wherein Ar¹, Ar², or both Ar¹ and Ar², are substituted with alkyl,alkoxy, aminocarbonyl, halogen, or alkyl-substituted thiol.
 4. Thecompound of claim 2, wherein Ar¹, Ar², or both Ar¹ and Ar², aresubstituted with alkyl, alkoxy, aminocarbonyl, halogen, oralkyl-substituted thiol.
 5. The compound of claim 1, wherein Ar¹, Ar²,or both Ar¹ and Ar², are optionally-substituted phenyl.
 6. Apharmaceutical composition comprising at least one compound of claim 1,and at least one pharmaceutically acceptable additive.